Albumin fusion proteins

ABSTRACT

The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disordrs or conditions using albumin fusion proteins of the invention.

This application claims the benefit of priority under 35 U.S.C. § 119(e)based on the following U.S. provisional applications: 60/229,358 filedon Apr. 12, 2000; 60/199,384 filed on Apr. 25, 2000; and 60/256,931filed on Dec. 21, 2000. Each of the provisional applications is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The invention relates generally to Therapeutic proteins (including, butnot limited to, a polypeptide, antibody, or peptide, or fragments andvariants thereof) fused to albumin or fragments or variants of albumin.The invention further relates to Therapeutic proteins (including, butnot limited to, a polypeptide, antibody, or peptide, or fragments andvariants thereof) fused to albumin or fragments or variants of albumin,that exhibit extended shelf-life and/or extended or therapeutic activityin solution. These fusion proteins are herein collectively referred toas “albumin fusion proteins of the invention.” The invention encompassestherapeutic albumin fusion proteins, compositions, pharmaceuticalcompositions, formulations and kits. Nucleic acid molecules encoding thealbumin fusion proteins of the invention are also encompassed by theinvention, as are vectors containing these nucleic acids, host cellstransformed with these nucleic acids vectors, and methods of making thealbumin fusion proteins of the invention using these nucleic acids,vectors, and/or host cells.

The invention is also directed to methods of in vitro stabilizing aTherapeutic protein via fusion or conjugation of the Therapeutic proteinto albumin or fragments or variants of albumin.

Human serum albumin (HSA, or HA), a protein of 585 amino acids in itsmature form (as shown in FIG. 15 or in SEQ ID NO:18), is responsible fora significant proportion of the osmotic pressure of serum and alsofunctions as a carrier of endogenous and exogenous ligands. At present,HA for clinical use is produced by extraction from human blood. Theproduction of recombinant HA (rHA) in microorganisms has been disclosedin EP 330 451 and EP 361 991.

The role of albumin as a carrier molecule and its inert nature aredesirable properties for use as a carrier and transporter ofpolypeptides in vivo. The use of albumin as a component of an albuminfusion protein as a carrier for various proteins has been suggested inWO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminalfragments of HA

This application claims the benefit of priority under 35 U.S.C. § 119(e)based on the following U.S., provisional applications: 60/229,358 filedon Apr. 12, 2000; 60/199,384 filed on Apr. 25, 2000; and 60/256,931filed on Dec. 21, 2000. Each of the provisional applications is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The invention relates generally to Therapeutic proteins (including, butnot limited to, a polypeptide, antibody, or peptide, or fragments andvariants thereof) fused to albumin or fragments or variants of albumin.The invention further relates to Therapeutic proteins (including, butnot limited to, a polypeptide, antibody, or peptide, or fragments andvariants thereof) fused to albumin or fragments or variants of albumin,that exhibit extended shelf-life and/or extended or therapeutic activityin solution. These fusion proteins are herein collectively referred toas “albumin fusion proteins of the invention.” The invention encompassestherapeutic albumin fusion proteins, compositions, pharmaceuticalcompositions, formulations and kits. Nucleic acid molecules encoding thealbumin fusion proteins of the invention are also encompassed by theinvention, as are vectors containing these nucleic acids, host cellstransformed with these nucleic acids vectors, and methods of making thealbumin fusion proteins of the invention using these nucleic acids,vectors, and/or host cells.

The invention is also directed to methods of in vitro stabilizing aTherapeutic protein via fusion or conjugation of the Therapeutic proteinto albumin or fragments or variants of albumin.

Human serum albumin (HSA, or HA), a protein of 585 amino acids in itsmature form (as shown in FIG. 15 or in SEQ ID NO:18), is responsible fora significant proportion of the osmotic pressure of serum and alsofunctions as a carrier of endogenous and exogenous ligands. At present,HA for clinical use is produced by extraction from human blood. Theproduction of recombinant. HA (rHA) in microorganisms has been disclosedin EP 330 451 and EP 361 991.

The role of albumin as a carrier molecule and its inert nature aredesirable properties for use as a carrier and transporter ofpolypeptides in vivo. The use of albumin as a component of an albuminfusion protein as a carrier for various proteins has been suggested inWO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminalfragments of HA for fusions to polypeptides has also been proposed (EP399 666). Fusion of albumin to the Therapeutic protein may be achievedby genetic manipulation, such that the DNA coding for HA, or a fragmentthereof, is joined to the DNA coding for the Therapeutic protein. Asuitable host is then transformed or transfected with the fusednucleotide sequences, so arranged on a suitable plasmid as to express afusion polypeptide. The expression may be effected in vitro from, forexample, prokaryotic or eukaryotic cells, or in vivo e.g. from atransgenic organism.

Therapeutic proteins in their native state or when recombinantlyproduced, such as interferons and growth hormones, are typically labilemolecules exhibiting short shelf-lives, particularly when formulated inaqueous solutions. The instability in these molecules when formulatedfor administration dictates that many of the molecules must belyophilized and refrigerated at all times during storage, therebyrendering the molecules difficult to transport and/or store. Storageproblems are particularly acute when pharmaceutical formulations must bestored and dispensed outside of the hospital environment. Many proteinand peptide drugs also require the addition of high concentrations ofother protein such as albumin to reduce or prevent loss of protein dueto binding to the container. This is a major concern with respect toproteins such as IFN. For this reason, many Therapeutic proteins areformulated in combination with large proportion of albumin carriermolecule (100-1000 fold excess), though this is an undesirable andexpensive feature of the formulation.

Few practical solutions to the storage problems of labile proteinmolecules have been proposed. Accordingly, there is a need forstabilized, long lasting formulations of proteinaceous therapeuticmolecules that are easily dispensed, preferably with a simpleformulation requiring minimal post-storage manipulation.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatTherapeutic proteins may be stabilized to extend the shelf-life, and/orto retain the Therapeutic protein's activity for extended periods oftime in solution, in vitro and/or in vivo, by genetically or chemicallyfusing or conjugating the Therapeutic protein to albumin or a fragment(portion) or variant of albumin, that is sufficient to stabilize theprotein and/or its activity. In addition it has been determined that theuse of albumin-fusion proteins or albumin conjugated proteins may reducethe need to formulate protein solutions with large excesses of carrierproteins (such as albumin, unfused) to prevent loss of Therapeuticproteins due to factors such as binding to the container.

The present invention encompasses albumin fusion proteins comprising aTherapeutic protein (e.g., a polypeptide, antibody, or peptide, orfragments and variants thereof) fused to albumin or a fragment (portion)or variant of albumin. The present invention also encompasses albuminfusion proteins comprising a Therapeutic protein (e.g., a polypeptide,antibody, or peptide, or fragments and variants thereof) fused toalbumin or a fragment (portion) or variant of albumin, that issufficient to prolong the shelf life of the Therapeutic protein, and/orstabilize the Therapeutic protein and/or its activity in solution (or ina pharmaceutical composition) in vitro and/or in vivo. Nucleic acidmolecules encoding the albumin fusion proteins of the invention are alsoencompassed by the invention, as are vectors containing these nucleicacids, host cells transformed with these nucleic acids vectors, andmethods of making the albumin fusion proteins of the invention and usingthese nucleic acids, vectors, and/or host cells.

The invention also encompasses pharmaceutical formulations comprising analbumin fusion protein of the invention and a pharmaceuticallyacceptable diluent or carrier. Such formulations may be in a kit orcontainer. Such kit or container may be packaged with instructionspertaining to the extended shelf life of the Therapeutic protein. Suchformulations may be used in methods of treating, preventing,ameliorating, or diagnosing a disease or disease symptom in a patient,preferably a mammal, most preferably a human, comprising the step ofadministering the pharmaceutical formulation to the patient.

In other embodiments, the present invention encompasses methods ofpreventing treating, or ameliorating a disease or disorder. In preferredembodiments, the present invention encompasses a method of treating adisease or disorder listed in the “Preferred Indication Y” column ofTable 1 comprising administering to a patient in which such treatment,prevention or amelioration is desired an albumin fusion protein of theinvention that comprises a Therapeutic protein portion corresponding toa Therapeutic protein (or fragment or variant thereof) disclosed in the“Therapeutic Protein X” column of Table 1 (in the same row as thedisease or disorder to be treated is listed in the “Preferred IndicationY” column of Table 1) in an amount effective to treat prevent orameliorate the disease or disorder.

In another embodiment, the invention includes a method of extending theshelf life of a Therapeutic protein (e.g., a polypeptide, antibody, orpeptide, or fragments and variants thereof) comprising the step offusing or conjugating the Therapeutic protein to albumin or a fragment(portion) or variant of albumin, that is sufficient to extend theshelf-life of the Therapeutic protein. In a preferred embodiment, theTherapeutic protein used according to this method is fused to thealbumin, or the fragment or variant of albumin. In a most preferredembodiment, the Therapeutic protein used according to this method isfused to albumin, or a fragment or variant of albumin, via recombinantDNA technology or genetic engineering.

In another embodiment, the invention includes a method of stabilizing aTherapeutic protein (e.g., a polypeptide, antibody, or peptide, orfragments and variants thereof) in solution, comprising the step offusing or conjugating the Therapeutic protein to albumin or a fragment(portion) or variant of albumin, that is sufficient to stabilize theTherapeutic protein. In a preferred embodiment, the Therapeutic proteinused according to this method is fused to the albumin, or the fragmentor variant of albumin. In a most preferred embodiment, the Therapeuticprotein used according to this method is fused to albumin, or a fragmentor variant of albumin, via recombinant DNA technology or geneticengineering.

The present invention further includes transgenic organisms modified tocontain the nucleic acid molecules of the invention, preferably modifiedto express the albumin fusion proteins encoded by the nucleic acidmolecules.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the extended shelf-life of an HA fusion protein in termsof the biological activity (Nb2 cell proliferation) of HA-hGH remainingafter incubation in cell culture media for up to 5 weeks at 37° C. Underthese conditions, hGH has no observed activity by week 2.

FIG. 2 depicts the extended shelf-life of an HA fusion protein in termsof the stable biological activity (Nb2 cell proliferation) of HA-hGHremaining after incubation in cell culture media for up to 3 weeks at 4,37, or 50° C. Data is normalized to the biological activity of hGH attime zero.

FIGS. 3A and 3B compare the biological activity of HA-hGH with hGH inthe Nb2 cell proliferation assay. FIG. 3A shows proliferation after 24hours of incubation with various concentrations of hGH or the albuminfusion protein, and FIG. 3B shows proliferation after 48 hours ofincubation with various concentrations of hGH or the albumin fusionprotein.

FIG. 4 shows a map of a plasmid (pPPC0005) that can be used as the basevector into which polynucleotides encoding the Therapeutic proteins(including polypeptide and fragments and variants thereof) may be clonedto form HA-fusions. Plasmid Map key: PRB1p: PRB1 S. cerevisiae promoter;FL: Fusion leader sequence; rHA: cDNA encoding HA, ADH1t: ADH1 S.cerevisiae terminator; T3: T3 sequencing primer site; T7: T7 sequencingprimer site; Amp R: β-lactamase gene; ori: origin of replication. Pleasenote that in the provisional applications to which this applicationclaims priority, the plasmid in FIG. 4 was labeled pPPC0006, instead ofpPPC0005. In addition the drawing of this plasmid did not show certainpertinent restriction sites in this vector. Thus in the presentapplication, the drawing is labeled pPPC0005 and more restriction sitesof the same vector are shown.

FIG. 5 compares the recovery of vial-stored HA-IFN solutions of variousconcentrations with a stock solution after 48 or 72 hours of storage.

FIG. 6 compares the activity of an HA-α-IFN fusion protein afteradministration to monkeys via IV or SC.

FIG. 7 describes the bioavailability and stability of an HA-α-IFN fusionprotein.

FIG. 8 is a map of an expression vector for the production of HA-_-IFN.

FIG. 9 shows the location of loops in HA.

FIG. 10 is an example of the modification of an HA loop.

FIG. 11 is a representation of the HA loops.

FIG. 12 shows the HA loop IV.

FIG. 13 shows the tertiary structure of HA.

FIG. 14 shows an example of a scFv-HA fusion

FIG. 15 shows the amino acid sequence of the mature form of humanalbumin (SEQ ID NO:18) and a polynucleotide encoding it (SEQ ID NO:17).

DETAILED DESCRIPTION

As described above, the present invention is based, in part, on thediscovery that a Therapeutic protein (e.g., a polypeptide, antibody, orpeptide, or fragments and variants thereof) may be stabilized to extendthe shelf-life and/or retain the Therapeutic protein's activity forextended periods of time in solution (or in a pharmaceuticalcomposition) in vitro and/or in vivo, by genetically fusing orchemically conjugating the Therapeutic protein, polypeptide or peptideto all or a portion of albumin sufficient to stabilize the protein andits activity.

The present invention relates generally to albumin fusion proteins andmethods of treating, preventing, or ameliorating diseases or disorders.As used herein, “albumin fusion protein” refers to a protein formed bythe fusion of at least one molecule of albumin (or a fragment or variantthereof) to at least one molecule of a Therapeutic protein (or fragmentor variant thereof). An albumin fusion protein of the inventioncomprises at least a fragment or variant of a Therapeutic protein and atleast a fragment or variant of human serum albumin, which are associatedwith one another, preferably by genetic fusion (i.e., the albumin fusionprotein is generated by translation of a nucleic acid in which apolynucleotide encoding all or a portion of a Therapeutic protein isjoined in-frame with a polynucleotide encoding all or a portion ofalbumin) or chemical conjugation to one another. The Therapeutic proteinand albumin protein, once part of the albumin fusion protein, may bereferred to as a “portion”, “region” or “moiety” of the albumin fusionprotein (e.g., a “Therapeutic protein portion” or an “albumin proteinportion”).

In one embodiment, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a Therapeutic protein (e.g.,as described in Table 1) and a serum albumin protein. In otherembodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a biologically active and/ortherapeutically active fragment of a Therapeutic protein and a serumalbumin protein. In other embodiments, the invention provides an albuminfusion protein comprising, or alternatively consisting of, abiologically active and/or therapeutically active variant of aTherapeutic protein and a serum albumin protein. In preferredembodiments, the serum albumin protein component of the albumin fusionprotein is the mature portion of serum albumin.

In further embodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a Therapeutic protein, and abiologically active and/or therapeutically active fragment of serumalbumin. In further embodiments, the invention provides an albuminfusion protein comprising, or alternatively consisting of, a Therapeuticprotein and a biologically active and/or therapeutically active variantof serum albumin. In preferred embodiments, the Therapeutic proteinportion of the albumin fusion protein is the mature portion of theTherapeutic protein. In a further preferred embodiment, the Therapeuticprotein portion of the albumin fusion protein is the extracellularsoluble domain of the Therapeutic protein. In an alternative embodiment,the Therapeutic protein portion of the albumin fusion protein is theactive form of the Therapeutic protein.

In further embodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a biologically active and/ortherapeutically active fragment or variant of a Therapeutic protein anda biologically active and/or therapeutically active fragment or variantof serum albumin. In preferred embodiments, the invention provides analbumin fusion protein comprising, or alternatively consisting of, themature portion of a Therapeutic protein and the mature portion of serumalbumin.

Therapeutic Proteins

As stated above, an albumin fusion protein of the invention comprises atleast a fragment or variant of a Therapeutic protein and at least afragment or variant of human serum albumin, which are associated withone another, preferably by genetic fusion or chemical conjugation.

As used herein, “Therapeutic protein” refers to proteins, polypeptides,antibodies, peptides or fragments or variants thereof, having one ormore therapeutic and/or biological activities. Therapeutic proteinsencompassed by the invention include but are not limited to, proteins,polypeptides, peptides, antibodies, and biologics. (The terms peptides,proteins, and polypeptides are used interchangeably herein.) It isspecifically contemplated that the term “Therapeutic protein”encompasses antibodies and fragments and variants thereof. Thus analbumin fusion protein of the invention may contain at least a fragmentor variant of a Therapeutic protein, and/or at least a fragment orvariant of an antibody. Additionally, the term “Therapeutic protein” mayrefer to the endogenous or naturally occurring correlate of aTherapeutic protein.

By a polypeptide displaying a “therapeutic activity” or a protein thatis “therapeutically active” is meant a polypeptide that possesses one ormore known biological and/or therapeutic activities associated with aTherapeutic protein such as one or more of the Therapeutic proteinsdescribed herein or otherwise known in the art. As a non-limitingexample, a “Therapeutic protein” is a protein that is useful to treat,prevent or ameliorate a disease, condition or disorder. As anon-limiting example, a “Therapeutic protein” may be one that bindsspecifically to a particular cell type (normal (e.g., lymphocytes) orabnormal e.g., (cancer cells)) and therefore may be used to target acompound (drug, or cytotoxic agent) to that cell type specifically.

In another non-limiting example, a “Therapeutic protein” is a proteinthat has a biological activity, and in particular, a biological activitythat is useful for treating preventing or ameliorating a disease. Anon-inclusive list of biological activities that may be possessed by aTherapeutic protein includes, enhancing the immune response, promotingangiogenesis, inhibiting angiogenesis, regulating hematopoieticfunctions, stimulating nerve growth, enhancing an immune response,inhibiting an immune response, or any one or more of the biologicalactivities described in the “Biological Activities” section below.

As used herein, “therapeutic activity” or “activity” may refer to anactivity whose effect is consistent with a desirable therapeutic outcomein humans, or to desired effects in non-human mammals or in otherspecies or organisms. Therapeutic activity may be measured in vivo or invitro. For example, a desirable effect may be assayed in cell culture.As an example, when hGH is the Therapeutic protein, the effects of hGHon cell proliferation as described in Example 1 may be used as theendpoint for which therapeutic activity is measured. Such in vitro orcell culture assays are commonly available for many Therapeutic proteinsas described in the art. Examples of assays include, but are not limitedto those described herein in the Examples section or in the “ExemplaryActivity Assay” column of Table 1.

Therapeutic proteins corresponding to a Therapeutic protein portion ofan albumin fusion protein of the invention, such as cell surface andsecretory proteins, are often modified by the attachment of one or moreoligosaccharide groups. The modification, referred to as glycosylation,can dramatically affect the physical properties of proteins and can beimportant in protein stability, secretion, and localization.Glycosylation occurs at specific locations along the polypeptidebackbone. There are usually two major types of glycosylation:glycosylation characterized by O-linked oligosaccharides, which areattached to serine or threonine residues; and glycosylationcharacterized by N-linked oligosaccharides, which are attached toasparagine residues in an Asn-X-Ser/Thr sequence, where X can be anyamino acid except proline. N-acetylneuramic acid (also known as sialicacid) is usually the terminal residue of both N-linked and 0-linkedoligosaccharides. Variables such as protein structure and cell typeinfluence the number and nature of the carbohydrate units within thechains at different glycosylation sites. Glycosylation isomers are alsocommon at the same site within a given cell type.

For example, several types of human interferon are glycosylated. Naturalhuman interferon-α2 is O-glycosylated at threonine 106, andN-glycosylation occurs at asparagine 72 in interferon-α14 (Adolf et al.,J. Biochem 276:511 (1991); Nyman T A et al., J. Biochem 329:295 (1998)).The oligosaccharides at asparagine 80 in natural interferon-β1α may playan important factor in the solubility and stability of the protein, butmay not be essential for its biological activity. This permits theproduction of an unglycosylated analog (interferon-β1b) engineered withsequence modifications to enhance stability (Hosoi et al., J. InterferonRes. 8:375 (1988; Karpusas et al., Cell Mol Life Sci 54:1203 (1998);Knight, J. Interferon Res. 2:421 (1982); Runkel et al., Pharm Res 15:641(1998); Lin, Dev. Biol. Stand. 96:97 (1998))1. Interferon-γ contains twoN-linked oligosaccharide chains at positions 25 and 97, both importantfor the efficient formation of the bioactive recombinant protein, andhaving an influence on the pharmacokinetic properties of the protein(Sareneva et al., Eur. J. Biochem 242:191 (1996); Sareneva et al.,Biochem J. 303:831 (1994); Sareneva et al., J. Interferon Res. 13:267(1993)). Mixed O-linked and N-linked glycosylation also occurs, forexample in human erythropoietin, N-linked glycosylation occurs atasparagine residues located at positions 24, 38 and 83 while O-linkedglycosylation occurs at a serine residue located at position 126 (Lai etal., J. Biol. Chem. 261:3116 (1986); Broudy et al., Arch. Biochem.Biophys. 265:329 (1988)).

Therapeutic proteins corresponding to a Therapeutic protein portion ofan albumin fusion protein of the invention, as well as analogs andvariants thereof, may be modified so that glycosylation at one or moresites is altered as a result of manipulation(s) of their nucleic acidsequence, by the host cell in which they are expressed, or due to otherconditions of their expression. For example, glycosylation isomers maybe produced by abolishing or introducing glycosylation sites, e.g., bysubstitution or deletion of amino acid residues, such as substitution ofglutamine for asparagine, or unglycosylated recombinant proteins may beproduced by expressing the proteins in host cells that will notglycosylate them, e.g. in E. coli or glycosylation-deficient yeast.These approaches are described in more detail below and are known in theart.

Therapeutic proteins (particularly those disclosed in Table 1) and theirnucleic acid sequences are well known in the art and available in publicdatabases such as Chemical Abstracts Services Databases (e.g., the CASRegistry), GenBank, and GenSeq as shown in Table 1.

Additional Therapeutic proteins corresponding to a Therapeutic proteinportion of an albumin fusion protein of the invention include, but arenot limited to, one or more of the Therapeutic proteins or peptidesdisclosed in the “Therapeutic Protein X” column of Table 1, or fragmentor variable thereof.

Table 1 provides a non-exhaustive list of Therapeutic proteins thatcorrespond to a Therapeutic protein portion of an albumin fusion proteinof the invention. The “Therapeutic Protein X” column disclosesTherapeutic protein molecules followed by parentheses containingscientific and brand names that comprise, or alternatively consist of,that Therapeutic protein molecule or a fragment or variant thereof.“Therapeutic protein X” as used herein may refer either to an individualTherapeutic protein molecule (as defined by the amino acid sequenceobtainable from the CAS and Genbank accession numbers), or to the entiregroup of Therapeutic proteins associated with a given Therapeuticprotein molecule disclosed in this column. The “Exemplary Identifier”column provides Chemical Abstracts Services (CAS) Registry Numbers(published by the American Chemical Society) and/or Genbank AccessionNumbers ((e.g., Locus ID, NP_XXXXX (Reference Sequence Protein), andXP_XXXXX (Model Protein) identifiers available through the nationalCenter for Biotechnology Information (NCBI) webpage atwww.ncbi.nlm.nih.gov) that correspond to entries in the CAS Registry orGenbank database which contain an amino acid sequence of the TherapeuticProtein Molecule or of a fragment or variant of the Therapeutic ProteinMolecule. In addition GenSeq Accession numbers and/or journalpublication citations are given to identify the exemplary amino acidsequence for some polypeptides. The summary pages associated with eachof these CAS and Genbank and GenSeq Accession Numbers as well as thecited journal publications (e.g., PubMed ID number (PMID)) are eachincorporated by reference in their entireties, particularly with respectto the amino acid sequences described therein. The “PCT/PatentReference” column provides U.S. patent numbers, or PCT InternationalPublication Numbers corresponding to patents and/or published patentapplications that describe the Therapeutic protein molecule. Each of thepatents and/or published patent applications cited in the “PCT/PatentReference” column are herein incorporated by reference in theirentireties. In particular, the amino acid sequences of the specifiedpolypeptide set forth in the sequence listing of each cited “PCT/PatentReference”, the variants of these amino acid sequences (mutations,fragments, etc.) set forth, for example, in the detailed description ofeach cited “PCT/Patent Reference”, the therapeutic indications setforth, for example, in the detailed description of each cited“PCT/Patent Reference”, and the activity asssays for the specifiedpolypeptide set forth in the detailed description, and moreparticularly, the examples of each cited “PCT/Patent Reference” areincorporated herein by reference. The “Biological activity” columndescribes Biological activities associated with the Therapeutic proteinmolecule. The “Exemplary Activity Assay” column provides references thatdescribe assays which may be used to test the therapeutic and/orbiological activity of a Therapeutic protein or an albumin fusionprotein of the invention comprising a Therapeutic protein X portion.Each of the references cited in the “Exemplary Activity Assay” columnare herein incorporated by reference in their entireties, particularlywith respect to the description of the respective activity assaydescribed in the reference (see Methods section, for example) forassaying the corresponding biological activity set forth in the“Biological Activity” column of Table 1. The “Preferred Indication Y”column describes disease, disorders, and/or conditions that may betreated, prevented, diagnosed, or ameliorated by Therapeutic protein Xor an albumin fusion protein of the invention comprising a Therapeuticprotein X portion. LENGTHY TABLE REFERENCED HEREUS20070026013A1-20070201-T00001 Please refer to the end of thespecification for access instructions.

In preferred embodiments, the albumin fusion proteins of the inventionare capable of a therapeutic activity and/or biologic activitycorresponding to the therapeutic activity and/or biologic activity ofthe Therapeutic protein corresponding to the Therapeutic protein portionof the albumin fusion protein listed in the corresponding row ofTable 1. (See, e.g., the “Biological Activity” and “Therapeutic ProteinX” columns of Table 1.) In further preferred embodiments, thetherapeutically active protein portions of the albumin fusion proteinsof the invention are fragments or variants of the reference sequencecited in the “Exemplary Identifier” column of Table 1, and are capableof the therapeutic activity and/or biologic activity of thecorresponding Therapeutic protein disclosed in “Biological Activity”column of Table 1.

Polypeptide and Polynucleotide Fragments and Variants

Fragments

The present invention is further directed to fragments of theTherapeutic proteins described in Table 1, albumin proteins, and/oralbumin fusion proteins of the invention.

Even if deletion of one or more amino acids from the N-terminus of aprotein results in modification or loss of one or more biologicalfunctions of the Therapeutic protein, albumin protein, and/or albuminfusion protein, other Therapeutic activities and/or functionalactivities (e.g., biological activities, ability to multimerize, abilityto bind a ligand) may still be retained. For example, the ability ofpolypeptides with N-terminal deletions to induce and/or bind toantibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a mutein with a large numberof deleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixamino acid residues may often evoke an immune response.

Accordingly, fragments of a Therapeutic protein corresponding to aTherapeutic protein portion of an albumin fusion protein of theinvention, include the full length protein as well as polypeptideshaving one or more residues deleted from the amino terminus of the aminoacid sequence of the reference polypeptide (e.g., a Therapeutic proteinas disclosed in Table 1). In particular, N-terminal deletions may bedescribed by the general formula m-q, where q is a whole integerrepresenting the total number of amino acid residues in a referencepolypeptide (e.g., a Therapeutic protein referred to in Table 1), and mis defined as any integer ranging from 2 to q-6. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

In addition, fragments of serum albumin polypeptides corresponding to analbumin protein portion of an albumin fusion protein of the invention,include the full length protein as well as polypeptides having one ormore residues deleted from the amino terminus of the amino acid sequenceof the reference polypeptide (i.e., serum albumin). In particular,N-terminal deletions may be described by the general formula m-585,where 585 is a whole integer representing the total number of amino acidresidues in serum albumin (SEQ ID NO:18), and m is defined as anyinteger ranging from 2 to 579. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Moreover, fragments of albumin fusion proteins of the invention, includethe full length albumin fusion protein as well as polypeptides havingone or more residues deleted from the amino terminus of the albuminfusion protein. In particular, N-terminal deletions may be described bythe general formula m-q, where q is a whole integer representing thetotal number of amino acid residues in the albumin fusion protein, and mis defined as any integer ranging from 2 to q-6. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the N-terminus or C-terminus of a reference polypeptide (e.g., aTherapeutic protein and/or serum albumin protein) results inmodification or loss of one or more biological functions of the protein,other functional activities (e.g., biological activities, ability tomultimerize, ability to bind a ligand) and/or Therapeutic activities maystill be retained. For example the ability of polypeptides withC-terminal deletions to induce and/or bind to antibodies which recognizethe complete or mature forms of the polypeptide generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking the N-terminal and/or C-terminal residues of areference polypeptide retains Therapeutic activity can readily bedetermined by routine methods described herein and/or otherwise known inthe art.

The present invention further provides polypeptides having one or moreresidues deleted from the carboxy terminus of the amino acid sequence ofa Therapeutic protein corresponding to a Therapeutic protein portion ofan albumin fusion protein of the invention (e.g., a Therapeutic proteinreferred to in Table 1). In particular, C-terminal deletions may bedescribed by the general formula 1−n, where n is any whole integerranging from 6 to q−1, and where q is a whole integer representing thetotal number of amino acid residues in a reference polypeptide (e.g., aTherapeutic protein referred to in Table 1). Polynucleotides encodingthese polypeptides are also encompassed by the invention.

In addition, the present invention provides polypeptides having one ormore residues deleted from the carboxy terminus of the amino acidsequence of an albumin protein corresponding to an albumin proteinportion of an albumin fusion protein of the invention (e.g., serumalbumin). In particular, C-terminal deletions may be described by thegeneral formula 1−n, where n is any whole integer ranging from 6 to 584,where 584 is the whole integer representing the total number of aminoacid residues in serum albumin (SEQ ID NO:18) minus 1. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Moreover, the present invention provides polypeptides having one or moreresidues deleted from the carboxy terminus of an albumin fusion proteinof the invention. In particular, C-terminal deletions may be describedby the general formula 1−n, where n is any whole integer ranging from 6to q−1, and where q is a whole integer representing the total number ofamino acid residues in an albumin fusion protein of the invention.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In addition, any of the above described N- or C-terminal deletions canbe combined to produce a N- and C-terminal deleted referencepolypeptide. The invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini, whichmay be described generally as having residues m-n of a referencepolypeptide (e.g., a Therapeutic protein referred to in Table 1, orserum albumin (e.g., SEQ ID NO:18), or an albumin fusion protein of theinvention) where n and m are integers as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identicalto a reference polypeptide sequence (e.g., a Therapeutic protein, serumalbumin protein or an albumin fusion protein of the invention) set forthherein, or fragments thereof. In preferred embodiments, the applicationis directed to proteins comprising polypeptides at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to reference polypeptides having theamino acid sequence of N- and C-terminal deletions as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Preferred polypeptide fragments of the invention are fragmentscomprising, or alternatively, consisting of, an amino acid sequence thatdisplays a Therapeutic activity and/or functional activity (e.g.biological activity) of the polypeptide sequence of the Therapeuticprotein or serum albumin protein of which the amino acid sequence is afragment. Other preferred polypeptide fragments are biologically activefragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thepolypeptide of the present invention. The biological activity of thefragments may include an improved desired activity, or a decreasedundesirable activity.

Variants

“Variant” refers to a polynucleotide or nucleic acid differing from areference nucleic acid or polypeptide, but retaining essentialproperties thereof. Generally, variants are overall closely similar,and, in many regions, identical to the reference nucleic acid orpolypeptide.

As used herein, “variant”, refers to a Therapeutic protein portion of analbumin fusion protein of the invention, albumin portion of an albuminfusion protein of the invention, or albumin fusion protein differing insequence from a Therapeutic protein (e.g. see “therapeutic” column ofTable 1), albumin protein, and/or albumin fusion protein of theinvention, respectively, but retaining at least one functional and/ortherapeutic property thereof (e.g., a therapeutic activity and/orbiological activity as disclosed in the “Biological Activity” column ofTable 1) as described elsewhere herein or otherwise known in the art.Generally, variants are overall very similar, and, in many regions,identical to the amino acid sequence of the Therapeutic proteincorresponding to a Therapeutic protein portion of an albumin fusionprotein of the invention, albumin protein corresponding to an albuminprotein portion of an albumin fusion protein of the invention, and/oralbumin fusion protein of the invention. Nucleic acids encoding thesevariants are also encompassed by the invention.

The present invention is also directed to proteins which comprise, oralternatively consist of, an amino acid sequence which is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example,the amino acid sequence of a Therapeutic protein corresponding to aTherapeutic protein portion of an albumin fusion protein of theinvention (e.g., an amino acid sequence disclosed in the “ExemplaryIdentifier” column of Table 1, or fragments or variants thereof),albumin proteins (e.g., SEQ ID NO:18 or fragments or variants thereof)corresponding to an albumin protein portion of an albumin fusion proteinof the invention, and/or albumin fusion proteins of the invention.Fragments of these polypeptides are also provided (e.g., those fragmentsdescribed herein). Further polypeptides encompassed by the invention arepolypeptides encoded by polynucleotides which hybridize to thecomplement of a nucleic acid molecule encoding an amino acid sequence ofthe invention under stringent hybridization conditions (e.g.,hybridization to filter bound DNA in 6× Sodium chloride/Sodium citrate(SSC) at about 45 degrees Celsius, followed by one or more washes in0.2×SSC, 0.1% SDS at about 50-65 degrees Celsius), under highlystringent conditions (e.g., hybridization to filter bound DNA in 6×sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius,followed by one or more washes in 0.1×SSC, 0.2% SDS at about 68 degreesCelsius), or under other stringent hybridization conditions which areknown to those of skill in the art (see, for example, Ausubel, F. M. etal., eds., 1989 Current protocol in Molecular Biology, Green publishingassociates, Inc., and John Wiley & Sons Inc., New York, at pages6.3.1-6.3.6 and 2.10.3). Polynucleotides encoding these polypeptides arealso encompassed by the invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino- or carboxy-terminal positions of the reference amino acidsequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence of an albumin fusion protein of the invention or afragment thereof (such as the Therapeutic protein portion of the albuminfusion protein or the albumin portion of the albumin fusion protein),can be determined conventionally using known computer programs. Apreferred method for determining the best overall match between a querysequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe FASTDB computer program based on the algorithm of Brutlag et al.(Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the queryand subject sequences are either both nucleotide sequences or both aminoacid sequences. The result of said global sequence alignment isexpressed as percent identity. Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The variant will usually have at least 75% (preferably at least about80%, 90%, 95% or 99%) sequence identity with a length of normal HA orTherapeutic protein which is the same length as the variant. Homology oridentity at the nucleotide or amino acid sequence level is determined byBLAST (Basic Local Alignment Search Tool) analysis using the algorithmemployed by the programs blastp, blastn, blastx, tblastn and tblastx(Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) andAltschul, J. Mol. Evol. 36: 290-300 (1993), fully incorporated byreference) which are tailored for sequence similarity searching.

The approach used by the BLAST program is to first consider similarsegments between a query sequence and a database sequence, then toevaluate the statistical significance of all matches that are identifiedand finally to summarize only those matches which satisfy a preselectedthreshold of significance. For a discussion of basic issues insimilarity searching of sequence databases, see Altschul et al., (NatureGenetics 6: 119-129 (1994)) which is fully incorporated by reference.The search parameters for histogram, descriptions, alignments, expect(i.e., the statistical significance threshold for reporting matchesagainst database sequences), cutoff, matrix and filter are at thedefault settings. The default scoring matrix used by blastp, blastx,tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., Proc.Natl. Acad. Sci. USA 89: 10915-10919 (1992), fully incorporated byreference). For blastn, the scoring matrix is set by the ratios of M(i.e., the reward score for a pair of matching residues) to N (i.e., thepenalty score for mismatching residues), wherein the default values forM and N are 5 and −4, respectively. Four blastn parameters may beadjusted as follows: Q=10 (gap creation penalty); R=10 (gap extensionpenalty); wink=1 (generates word hits at every wink^(th) position alongthe query); and gapw=16 (sets the window width within which gappedalignments are generated). The equivalent Blastp parameter settings wereQ=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences,available in the GCG package version 10.0, uses DNA parameters GAP=50(gap creation penalty) and LEN=3 (gap extension penalty) and theequivalent settings in protein comparisons are GAP=8 and LEN=2.

The polynucleotide variants of the invention may contain alterations inthe coding regions, non-coding regions, or both. Especially preferredare polynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. Nucleotide variants producedby silent substitutions due to the degeneracy of the genetic code arepreferred. Moreover, polypeptide variants in which less than 50, lessthan 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10,1-5, or 1-2 amino acids are substituted, deleted, or added in anycombination are also preferred. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host, such as, yeast or E. coli).

In a preferred embodiment, a polynucleotide encoding an albumin portionof an albumin fusion protein of the invention is optimized forexpression in yeast or mammalian cells. In further preferred embodiment,a polynucleotide encoding a Therapeutic protein portion of an albuminfusion protein of the invention is optimized for expression in yeast ormammalian cells. In a still further preferred embodiment, apolynucleotide encoding an albumin fusion protein of the invention isoptimized for expression in yeast or mammalian cells.

In an alternative embodiment, a codon optimized polynucleotide encodinga Therapeutic protein portion of an albumin fusion protein of theinvention does not hybridize to the wild type polynucleotide encodingthe Therapeutic protein under stringent hybridization conditions asdescribed herein. In a further embodiment, a codon optimizedpolynucleotide encoding an albumin portion of an albumin fusion proteinof the invention does not hybridize to the wild type polynucleotideencoding the albumin protein under stringent hybridization conditions asdescribed herein. In another embodiment, a codon optimizedpolynucleotide encoding an albumin fusion protein of the invention doesnot hybridize to the wild type polynucleotide encoding the Therapeuticprotein portin or the albumin protein portion under stringenthybridization conditions as described herein.

In an additional embodiment, polynucleotides encoding a Therapeuticprotein portion of an albumin fusion protein of the invention do notcomprise, or alternatively consist of, the naturally occurring sequenceof that Therapeutic protein. In a further embodiment, polynucleotidesencoding an albumin protein portion of an albumin fusion protein of theinvention do not comprise, or alternatively consist of, the naturallyoccurring sequence of albumin protein. In an alternative embodiment,polynucleotides encoding an albumin fusion protein of the invention donot comprise, or alternatively consist of, of the naturally occurringsequence of a Therapeutic protein portion or the albumin proteinportion.

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the polypeptide of the present invention withoutsubstantial loss of biological function. As an example, Ron et al. (J.Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins havingheparin binding activity even after deleting 3, 8, or 27 amino-terminalamino acid residues. Similarly, Interferon gamma exhibited up to tentimes higher activity after deleting 8-10 amino acid residues from thecarboxy terminus of this protein. (Dobeli et al., J. Biotechnology7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” In fact, only 23 unique amino acid sequences, outof more than 3,500 nucleotide sequences examined, produced a proteinthat significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which have afunctional activity (e.g., biological activity and/or therapeuticactivity). In highly preferred embodiments the invention providesvariants of albumin fusion proteins that have a functional activity(e.g., biological activity and/or therapeutic activity, such as thatdisclosed in the “Biological Activity” column in Table 1) thatcorresponds to one or more biological and/or therapeutic activities ofthe Therapeutic protein corresponding to the Therapeutic protein portionof the albumin fusion protein. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.

In preferred embodiments, the variants of the invention haveconservative substitutions. By “conservative substitutions” is intendedswaps within groups such as replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided, for example, in Bowie et al., “Decipheringthe Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main strategies for studying the toleranceof an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. See Cunningham and Wells,Science 244:1081-1085 (1989). The resulting mutant molecules can then betested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gin, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly. Besides conservative amino acid substitution, variants of thepresent invention include (i) polypeptides containing substitutions ofone or more of the non-conserved amino acid residues, where thesubstituted amino acid residues may or may not be one encoded by thegenetic code, or (ii) polypeptides containing substitutions of one ormore of the amino acid residues having a substituent group, or (iii)polypeptides which have been fused with or chemically conjugated toanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), (iv)polypeptide containing additional amino acids, such as, for example, anIgG Fc fusion region peptide, Such variant polypeptides are deemed to bewithin the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

In specific embodiments, the polypeptides of the invention comprise, oralternatively, consist of, fragments or variants of the amino acidsequence of a Therapeutic protein described herein and/or human serumalbumin, and/or albumin fusion protein of the invention, wherein thefragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, aminoacid residue additions, substitutions, and/or deletions when compared tothe reference amino acid sequence. In preferred embodiments, the aminoacid substitutions are conservative. Nucleic acids encoding thesepolypeptides are also encompassed by the invention.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR. PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992)).

Functional Activity

“A polypeptide having functional activity” refers to a polypeptidecapable of displaying one or more known functional activities associatedwith the full-length, pro-protein, and/or mature form of a Therapeuticprotein. Such functional activities include, but are not limited to,biological activity, antigenicity [ability to bind (or compete with apolypeptide for binding) to an anti-polypeptide antibody],immunogenicity (ability to generate antibody which binds to a specificpolypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide.

“A polypeptide having biological activity” refers to a polypeptideexhibiting activity similar to, but not necessarily identical to, anactivity of a Therapeutic protein of the present invention, includingmature forms, as measured in a particular biological assay, with orwithout dose dependency. In the case where dose dependency does exist,it need not be identical to that of the polypeptide, but rathersubstantially similar to the dose-dependence in a given activity ascompared to the polypeptide of the present invention (i.e., thecandidate polypeptide will exhibit greater activity or not more thanabout 25-fold less and, preferably, not more than about tenfold lessactivity, and most preferably, not more than about three-fold lessactivity relative to the polypeptide of the present invention).

In preferred embodiments, an albumin fusion protein of the invention hasat least one biological and/or therapeutic activity associated with theTherapeutic protein (or fragment or variant thereof) when it is notfused to albumin.

The albumin fusion proteins of the invention can be assayed forfunctional activity (e.g., biological activity) using or routinelymodifying assays known in the art, as well as assays described herein.Specifically, albumin fusion proteins may be assayed for functionalactivity (e.g., biological activity or therapeutic activity) using theassay referenced in the “Exemplary Activity Assay” column of Table 1.Additionally, one of skill in the art may routinely assay fragments of aTherapeutic protein corresponding to a Therapeutic protein portion of analbumin fusion protein of the invention, for activity using assaysreferenced in its corresponding row of Table 1. Further, one of skill inthe art may routinely assay fragments of an albumin proteincorresponding to an albumin protein portion of an albumin fusion proteinof the invention, for activity using assays known in the art and/or asdescribed in the Examples section below.

For example, in one embodiment where one is assaying for the ability ofan albumin fusion protein of the invention to bind or compete with aTherapeutic protein for binding to an anti-Therapeutic polypeptideantibody and/or anti-albumin antibody, various immunoassays known in theart can be used, including but not limited to, competitive andnon-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

In a preferred embodiment, where a binding partner (e.g., a receptor ora ligand) of a Therapeutic protein is identified, binding to thatbinding partner by an albumin fusion protein containing that Therapeuticprotein as the Therapeutic protein portion of the fusion can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky et al., Microbiol. Rev.59:94-123 (1995). In another embodiment, the ability of physiologicalcorrelates of an albumin fusion protein of the present invention to bindto a substrate(s) of the Therapeutic polypeptide corresponding to theTherapeutic portion of the albumin fusion protein of the invention canbe routinely assayed using techniques known in the art.

In an alternative embodiment, where the ability of an albumin fusionprotein of the invention to multimerize is being evaluated, associationwith other components of the multimer can be assayed, e.g., by meanswell-known in the art, such as, for example, reducing and non-reducinggel chromatography, protein affinity chromatography, and affinityblotting. See generally, Phizicky et al., supra.

In preferred embodiments, an albumin fusion protein of the inventioncomprising all or a portion of an antibody that binds a Therapeuticprotein, has at least one biological and/or therapeutic activity (e.g.,to specifically bind a polypeptide or epitope) associated with theantibody that binds a Therapeutic protein (or fragment or variantthereof) when it is not fused to albumin. In other preferredembodiments, the biological activity and/or therapeutic activity of analbumin fusion protein of the invention comprising all or a portion ofan antibody that binds a Therapeutic protein is the inhibition (i.e.antagonism) or activation (i.e., agonism) of one or more of thebiological activities and/or therapeutic activities associated with thepolypeptide that is specifically bound by antibody that binds aTherapeutic protein.

Albumin fusion proteins of the invention (e.g., comprising at least afragment or variant of an antibody that binds a Therapeutic protein) maybe characterized in a variety of ways. In particular, albumin fusionproteins of the invention comprising at least a fragment or variant ofan antibody that binds a Therapeutic protein may be assayed for theability to specifically bind to the same antigens specifically bound bythe antibody that binds a Therapeutic protein corresponding to theTherapeutic protein portion of the albumin fusion protein usingtechniques described herein or routinely modifying techniques known inthe art.

Assays for the ability of the albumin fusion proteins of the invention(e.g., comprising at least a fragment or variant of an antibody thatbinds a Therapeutic protein) to (specifically) bind a specific proteinor epitope may be performed in solution (e.g., Houghten, Bio/Techniques13:412-421(1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), onchips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S.Pat. No. 5,223,409), on spores (e.g., U.S. Pat. Nos. 5,571,698;5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl.Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith,Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirlaet al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J.Mol. Biol. 222:301-310 (1991)) (each of these references is incorporatedherein in its entirety by reference). Albumin fusion proteins of theinvention comprising at least a fragment or variant of a Therapeuticantibody may also be assayed for their specificity and affinity for aspecific protein or epitope using or routinely modifying techniquesdescribed herein or otherwise known in the art.

The albumin fusion proteins of the invention comprising at least afragment or variant of an antibody that binds a Therapeutic protein maybe assayed for cross-reactivity with other antigens (e.g., moleculesthat have sequence/structure conservation with the molecule(s)specifically bound by the antibody that binds a Therapeutic protein (orfragment or variant thereof) corresponding to the Therapeutic proteinportion of the albumin fusion protein of the invention) by any methodknown in the art.

Immunoassays which can be used to analyze (immunospecific) binding andcross-reactivity include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, and protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety). Exemplary immunoassays are described briefly below(but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the albumin fusion protein of the invention (e.g., comprising atleast a fragment or variant of an antibody that binds a Therapeuticprotein) to the cell lysate, incubating for a period of time (e.g., 1 to4 hours) at 40 degrees C., adding sepharose beads coupled to ananti-albumin antibody, for example, to the cell lysate, incubating forabout an hour or more at 40 degrees C., washing the beads in lysisbuffer and resuspending the beads in SDS/sample buffer. The ability ofthe albumin fusion protein of the invention to immunoprecipitate aparticular antigen can be assessed by, e.g., western blot analysis. Oneof skill in the art would be knowledgeable as to the parameters that canbe modified to increase the binding of the albumin fusion protein to anantigen and decrease the background (e.g., pre-clearing the cell lysatewith sepharose beads). For further discussion regardingimmunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), applying the albuminfusion protein of the invention (diluted in blocking buffer) to themembrane, washing the membrane in washing buffer, applying a secondaryantibody (which recognizes the albumin fusion protein, e.g., ananti-human serum albumin antibody) conjugated to an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) or radioactivemolecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing themembrane in wash buffer, and detecting the presence of the antigen. Oneof skill in the art would be knowledgeable as to the parameters that canbe modified to increase the signal detected and to reduce the backgroundnoise. For further discussion regarding western blot protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, washing away antigen that did notbind the wells, adding the albumin fusion protein (e.g., comprising atleast a fragment or variant of an antibody that binds a Therapeuticprotein) of the invention conjugated to a detectable compound such as anenzymatic substrate (e.g., horseradish peroxidase or alkalinephosphatase) to the wells and incubating for a period of time, washingaway unbound or non-specifically bound albumin fusion proteins, anddetecting the presence of the albumin fusion proteins specifically boundto the antigen coating the well. In ELISAs the albumin fusion proteindoes not have to be conjugated to a detectable compound; instead, asecond antibody (which recognizes albumin fusion protein) conjugated toa detectable compound may be added to the well. Further, instead ofcoating the well with the antigen, the albumin fusion protein may becoated to the well. In this case, the detectable molecule could be theantigen conjugated to a detectable compound such as an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase). One ofskill in the art would be knowledgeable as to the parameters that can bemodified to increase the signal detected as well as other variations ofELISAs known in the art. For further discussion regarding ELISAs see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an albumin fusion protein to a protein, antigen,or epitope and the off-rate of an albumin fusionprotein-protein/antigen/epitope interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., ³H or ¹²⁵I) with the albumin fusion protein of the invention inthe presence of increasing amounts of unlabeled antigen, and thedetection of the antibody bound to the labeled antigen. The affinity ofthe albumin fusion protein of the present invention for a specificprotein, antigen, or epitope and the binding off-rates can be determinedfrom the data by Scatchard plot analysis. Competition with a secondprotein that binds the same protein, antigen or epitope as the albuminfusion protein, can also be determined using radioimmunoassays. In thiscase, the protein, antigen or epitope is incubated with an albuminfusion protein of the present invention conjugated to a labeled compound(e.g., ³H or ¹²⁵) in the presence of increasing amounts of an unlabeledsecond protein that binds the same protein, antigen, or epitope as thealbumin fusion protein of the invention.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of albumin fusion proteins of the inventionto a protein, antigen or epitope. BIAcore kinetic analysis comprisesanalyzing the binding and dissociation of albumin fusion proteins, orspecific polypeptides, antigens or epitopes from chips with immobilizedspecific polypeptides, antigens or epitopes or albumin fusion proteins,respectively, on their surface.

Antibodies that bind a Therapeutic protein corresponding to theTherapeutic protein portion of an albumin fusion protein of theinvention may also be described or specified in terms of their bindingaffinity for a given protein or antigen, preferably the antigen whichthey specifically bind. Preferred binding affinities include those witha dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10−3 M, 10−3M, 5×10⁻⁴ M, 10⁻⁴ M. More preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M or 10⁻⁸ M. Even more preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.In preferred embodiments, albumin fusion proteins comprising at least afragment or variant of an antibody that binds a Therapeutic protein, hasan affinity for a given protein or epitope similar to that of thecorresponding antibody (not fused to albumin) that binds a Therapeuticprotein, taking into account the valency of the albumin fusion protein(comprising at least a fragment or variant of an antibody that binds aTherapeutic protein) and the valency of the corresponding antibody.

In addition, assays described herein (see Examples and Table 1) andotherwise known in the art may routinely be applied to measure theability of albumin fusion proteins of the present invention andfragments, variants and derivatives thereof to elicit biologicalactivity and/or Therapeutic activity (either in vitro or in vivo)related to either the Therapeutic protein portion and/or albumin portionof the albumin fusion protein of the present invention. Other methodswill be known to the skilled artisan and are within the scope of theinvention.

Albumin

As described above, an albumin fusion protein of the invention comprisesat least a fragment or variant of a Therapeutic protein and at least afragment or variant of human serum albumin, which are associated withone another, preferably by genetic fusion or chemical conjugation.

The terms, human serum albumin (HSA) and human albumin (HA) are usedinterchangeably herein. The terms, “albumin and “serum albumin” arebroader, and encompass human serum albumin (and fragments and variantsthereof) as well as albumin from other species (and fragments andvariants thereof).

As used herein, “albumin” refers collectively to albumin protein oramino acid sequence, or an albumin fragment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular, “albumin” refers to human albumin or fragments thereof (seeEP 201 239, EP 322 094 WO 97/24445, WO95/23857) especially the matureform of human albumin as shown in FIG. 15 and SEQ ID NO: 18, or albuminfrom other vertebrates or fragments thereof, or analogs or variants ofthese molecules or fragments thereof.

In preferred embodiments, the human serum albumin protein used in thealbumin fusion proteins of the invention contains one or both of thefollowing sets of point mutations with reference to SEQ ID NO:18:Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala, and Arg-410 to Ala; orArg-410 to A, Lys-413 to Gln, and Lys-414 to Gln (see, e.g.,International Publication No. WO95/23857, hereby incorporated in itsentirety by reference herein). In even more preferred embodiments,albumin fusion proteins of the invention that contain one or both ofabove-described sets of point mutations have improvedstability/resistance to yeast Yap3p proteolytic cleavage, allowingincreased production of recombinant albumin fusion proteins expressed inyeast host cells.

As used herein, a portion of albumin sufficient to prolong thetherapeutic activity or shelf-life of the Therapeutic protein refers toa portion of albumin sufficient in length or structure to stabilize orprolong the therapeutic activity of the protein so that the shelf lifeof the Therapeutic protein portion of the albumin fusion protein isprolonged or extended compared to the shelf-life in the non-fusionstate. The albumin portion of the albumin fusion proteins may comprisethe full length of the HA sequence as described above or as shown inFIG. 15, or may include one or more fragments thereof that are capableof stabilizing or prolonging the therapeutic activity. Such fragmentsmay be of 10 or more amino acids in length or may include about 15, 20,25, 30, 50; or more contiguous amino acids from the HA sequence or mayinclude part or all of specific domains of HA. For instance, one or morefragments of HA spanning the first two immunoglobulin-like domains maybe used.

The albumin portion of the albumin fusion proteins of the invention maybe a variant of normal HA. The Therapeutic protein portion of thealbumin fusion proteins of the invention may also be variants of theTherapeutic proteins as described herein. The term “variants” includesinsertions, deletions and substitutions, either conservative or nonconservative, where such changes do not substantially alter one or moreof the oncotic, useful ligand-binding and non-immunogenic properties ofalbumin, or the active site, or active domain which confers thetherapeutic activities of the Therapeutic proteins.

In particular, the albumin fusion proteins of the invention may includenaturally occurring polymorphic variants of human albumin and fragmentsof human albumin, for example those fragments disclosed in EP 322 094(namely HA (Pn), where n is 369 to 419). The albumin may be derived fromany vertebrate, especially any mammal, for example human, cow, sheep, orpig. Non-mammalian albumins include, but are not limited to, hen andsalmon. The albumin portion of the albumin fusion protein may be from adifferent animal than the Therapeutic protein portion.

Generally speaking, an HA fragment or variant will be at least 100 aminoacids long, preferably at least 150 amino acids long. The HA variant mayconsist of or alternatively comprise at least one whole domain of HA,for example domains 1 (amino acids 1-194 of SEQ ID NO: 18), 2 (aminoacids 195-387 of SEQ ID NO: 18), 3 (amino acids 388-585 of SEQ IDNO:18), 1+2 (1-387 of SEQ ID NO:18), 2+3 (195-585 of SEQ ID NO:18) or1+3 (amino acids 1-194 of SEQ ID NO:18+amino acids 388-585 of SEQ IDNO:18). Each domain is itself made up of two homologous subdomainsnamely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, withflexible inter-subdomain linker regions comprising residues Lys106 toGlu119, Glu292 to Val315 and Glu492 to Ala511.

Preferably, the albumin portion of an albumin fusion protein of theinvention comprises at least one subdomain or domain of HA orconservative modifications thereof. If the fusion is based onsubdomains, some or all of the adjacent linker is preferably used tolink to the Therapeutic protein moiety.

Antibodies that Specifically Bind Therapeutic Proteins are AlsoTherapeutic Proteins

The present invention also encompasses albumin fusion proteins thatcomprise at least a fragment or variant of an antibody that specificallybinds a Therapeutic protein disclosed in Table 1. It is specificallycontemplated that the term “Therapeutic protein” encompasses antibodiesthat bind a Therapeutic protein and fragments and variants thereof. Thusan albumin fusion protein of the invention may contain at least afragment or variant of a Therapeutic protein, and/or at least a fragmentor variant of an an antibody that binds a Therapeutic protein.

Antibody Structure and Background

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Seegenerally, Fundamental Immunology Chapters 3-5 (Paul, W., ed., 4th ed.Raven Press, N.Y. (1998)) (incorporated by reference in its entirety forall purposes). The variable regions of each light/heavy chain pair formthe antibody binding site.

Thus, an intact IgG antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRregions, in general, are the portions of the antibody which make contactwith the antigen and determine its specificity. The CDRs from the heavyand the light chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains variable regions comprise the domains FR1,CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions are connectedto the heavy or light chain constant region. The assignment of aminoacids to each domain is in accordance with the definitions of KabatSequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917(1987); Chothia et al. Nature 342:878-883 (1989).

As used herein, “antibody” refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsan antigen (e.g., a molecule containing one or more CDR regions of anantibody). Antibodies that may correspond to a Therapeutic proteinportion of an albumin fusion protein include, but are not limited to,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies (e.g., single chain Fvs), Fab fragments, F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesspecific to antibodies of the invention), and epitope-binding fragmentsof any of the above (e.g., VH domains, VL domains, or one or more CDRregions).

Antibodies that Bind Therapeutic Proteins

The present invention encompasses albumin fusion proteins that compriseat least a fragment or variant of an antibody that binds a TherapeuticProtein (e.g., as disclosed in Table 1) or fragment or variant thereof.

Antibodies that bind a Therapeutic protein (or fragment or variantthereof) may be from any animal origin, including birds and mammals.Preferably, the antibodies are human, murine (e.g., mouse and rat),donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chickenantibodies. Most preferably, the antibodies are human antibodies. Asused herein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries and xenomice or other organisms that havebeen genetically engineered to produce human antibodies.

The antibody molecules that bind to a Therapeutic protein and that maycorrespond to a Therapeutic protein portion of an albumin fusion proteinof the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA andIgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. In preferred embodiments, the antibodymolecules that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein of theinvention are IgG1. In other preferred embodiments, the immunoglobulinmolecules that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein of theinvention are IgG2. In other preferred embodiments, the immunoglobulinmolecules that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein of theinvention are IgG4.

Most preferably the antibodies that bind to a Therapeutic protein andthat may correspond to a Therapeutic protein portion of an albuminfusion protein of the invention are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains.

The antibodies that bind to a Therapeutic protein and that maycorrespond to a Therapeutic protein portion of an albumin fusion proteinof the invention may be monospecific, bispecific, trispecific or ofgreater multispecificity. Multispecific antibodies may be specific fordifferent epitopes of a Therapeutic protein or may be specific for botha Therapeutic protein as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies that bind a Therapeutic protein (or fragment or variantthereof) may be bispecific or bifunctional which means that the antibodyis an artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann Clin. Exp.Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol. 148:1547 1553(1992). In addition, bispecific antibodies may be formed as “diabodies”(Holliger et al. “‘Diabodies’: small bivalent and bispecific antibodyfragments” PNAS USA 90:6444-6448 (1993)) or “Janusins” (Traunecker etal. “Bispecific single chain molecules (Janusins) target cytotoxiclymphocytes on HIV infected cells” EMBO J. 10:3655-3659 (1991) andTraunecker et al. “Janusin: new molecular design for bispecificreagents” Int J Cancer Suppl 7:51-52 (1992)).

The present invention also provides albumin fusion proteins thatcomprise, fragments or variants (including derivatives) of an antibodydescribed herein or known elsewhere in the art. Standard techniquesknown to those of skill in the art can be used to introduce mutations inthe nucleotide sequence encoding a molecule of the invention, including,for example, site-directed mutagenesis and PCR-mediated mutagenesiswhich result in amino acid substitutions. Preferably, the variants(including derivatives) encode less than 50 amino acid substitutions,less than 40 amino acid subsitutions, less than 30 amino acidsubstitutions, less than 25 amino acid substitutions, less than 20 aminoacid substitutions, less than 15 amino acid substitutions, less than 10amino acid substitutions, less than 5 amino acid substitutions, lessthan 4 amino acid substitutions, less than 3 amino acid substitutions,or less than 2 amino acid substitutions relative to the reference VHdomain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. Inspecific embodiments, the variants encode substitutions of VHCDR3. In apreferred embodiment, the variants have conservative amino acidsubstitutions at one or more predicted non-essential amino acidresidues.

Antibodies that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein of theinvention may be described or specified in terms of the epitope(s) orportion(s) of a Therapeutic protein which they recognize or specificallybind. Antibodies which specifically bind a Therapeutic protein or aspecific epitope of a Therapeutic protein may also be excluded.Therefore, the present invention encompasses antibodies thatspecifically bind Therapeutic proteins, and allows for the exclusion ofthe same. In preferred embodiments, albumin fusion proteins comprisingat least a fragment or variant of an antibody that binds a Therapeuticprotein, binds the same epitopes as the corresponding antibody (notfused to albumin) that binds a Therapeutic protein.

Antibodies that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein of theinvention may also be described or specified in terms of theircross-reactivity. Antibodies that do not bind any other analog,ortholog, or homolog, of a Therapeutic protein are included. Antibodiesthat bind polypeptides with at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 65%, at least 60%, atleast 55%, and at least 50% identity (as calculated using methods knownin the art and described herein) to a Therapeutic protein are alsoincluded in the present invention. In specific embodiments, antibodiesthat bind to a Therapeutic protein and that may correspond to aTherapeutic protein portion of an albumin fusion protein of theinvention cross-react with murine, rat and/or rabbit homologs of humanproteins and the corresponding epitopes thereof. Antibodies that do notbind polypeptides with less than 95%, less than 90%, less than 85%, lessthan 80%, less than 75%, less than 70%, less than 65%, less than 60%,less than 55%, and less than 50% identity (as calculated using methodsknown in the art and described herein) to a Therapeutic protein are alsoincluded in the present invention. In a specific embodiment, theabove-described cross-reactivity is with respect to any single specificantigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5,or more of the specific antigenic and/or immunogenic polypeptidesdisclosed herein. In preferred embodiments, albumin fusion proteinscomprising at least a fragment or variant of an antibody that binds aTherapeutic protein, has similar or substantially identical crossreactivity characteristics compared to the corresponding antibody (notfused to albumin) that binds a Therapeutic protein.

Further included in the present invention are antibodies which bindpolypeptides encoded by polynucleotides which hybridize to apolynucleotide encoding a Therapeutic protein under stringenthybridization conditions (as described herein). Antibodies that bind toa Therapeutic protein and that may correspond to a Therapeutic proteinportion of an albumin fusion protein of the invention may also bedescribed or specified in terms of their binding affinity to apolypeptide of the invention. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M,10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M. More preferred binding affinities includethose with a dissociation constant or Kd less than 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M or 10⁻⁸ M. Even morepreferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10¹⁰⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10⁻¹⁵ M. In preferred embodiments, albumin fusion proteinscomprising at least a fragment or variant of an antibody that binds aTherapeutic protein, has an affinity for a given protein or epitopesimilar to that of the corresponding antibody (not fused to albumin)that binds a Therapeutic protein, taking into account the valency of thealbumin fusion protein (comprising at least a fragment or variant of anantibody that binds a Therapeutic protein) and the valency of thecorresponding antibody.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of a Therapeutic protein asdetermined by any method known in the art for determining competitivebinding, for example, the immunoassays described herein. In preferredembodiments, the antibody competitively inhibits binding to the epitopeby at least 95%, at least 90%, at least 85%, at least 80%, at least 75%,at least 70%, at least 60%, or at least 50%. In preferred embodiments,albumin fusion proteins comprising at least a fragment or variant of anantibody that binds a Therapeutic protein, competitively inhibitsbinding of an antibody to an epitope of a Therapeutic protein as well asthe corresponding antibody (not fused to albumin) that binds aTherapeutic protein, competitively inhibits binding of an antibody to anepitope of a Therapeutic protein. In other preferred embodiments,albumin fusion proteins comprising at least a fragment or variant of anantibody that binds a Therapeutic protein, competitively inhibitsbinding of the corresponding antibody (not fused to albumin) that bindsa Therapeutic protein to an epitope of a Therapeutic protein by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein of theinvention may act as agonists or antagonists of the Therapeutic protein.For example, the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. The invention features both receptor-specificantibodies and ligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody. In preferred embodiments, albumin fusion proteinscomprising at least a fragment or variant of an antibody that binds aTherapeutic protein, has similar or substantially similarcharacteristics with regard to preventing ligand binding and/orpreventing receptor activation compared to the corresponding antibody(not fused to albumin) that binds a Therapeutic protein.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the Therapeuticprotreins (e.g. as disclosed in Table 1). The above antibody agonistscan be made using methods known in the art. See, e.g., PCT publicationWO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988(1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al.,J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res.58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179(1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard etal., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al.,Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem.272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):1420 (1996) (which are all incorporated by referenceherein in their entireties). In preferred embodiments, albumin fusionproteins comprising at least a fragment or variant of an antibody thatbinds a Therapeutic protein, have similar or substantially identicalagonist or antagonist properties as the corresponding antibody thatbinds a Therapeutic protein not fused to albumin.

Antibodies that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein of theinvention may be used, for example, to purify, detect, and targetTherapeutic proteins, including both in in vitro and in vivo diagnosticand therapeutic methods. For example, the antibodies have utility inimmunoassays for qualitatively and quantitatively measuring levels ofthe Therapeutic protein in biological samples. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); incorporated by reference herein in its entirety.Likewise, albumin fusion proteins comprising at least a fragment orvariant of an antibody that binds a Therapeutic protein, may be used,for example, to purify, detect, and target Therapeutic proteins,including both in in vitro and in vivo diagnostic and therapeuticmethods.

Antibodies that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein includederivatives that are modified, i.e, by the covalent attachment of anytype of molecule to the antibody. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids. Albumin fusion proteins of the invention may also bemodified as described above.

Methods of Producing Antibodies that Bind Therapeutic Proteins

The antibodies that bind to a Therapeutic protein and that maycorrespond to a Therapeutic protein portion of an albumin fusion proteinof the invention may be generated by any suitable method known in theart. Polyclonal antibodies to an antigen-of-interest can be produced byvarious procedures well known in the art. For example, a Therapeuticprotein may be administered to various host animals including, but notlimited to, rabbits, mice, rats, etc. to induce the production of seracontaining polyclonal antibodies specific for the antigen. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with a Therapeutic proteinor fragment or variant thereof or a cell expressing such a Therapeuticprotein or fragment or variant thereof. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody wherein,preferably, the hybridoma is generated by fusing splenocytes isolatedfrom a mouse immunized with an antigen of the invention with myelomacells and then screening the hybridomas resulting from the fusion forhybridoma clones that secrete an antibody able to bind a polypeptide ofthe invention.

Another well known method for producing both polyclonal and monoclonalhuman B cell lines is transformation using Epstein Barr Virus (EBV).Protocols for generating EBV-transformed B cell lines are commonly knownin the art, such as, for example, the protocol outlined in Chapter 7.22of Current Protocols in Immunology, Coligan et al., Eds., 1994, JohnWiley & Sons, NY, which is hereby incorporated in its entirety byreference. The source of B cells for transformation is commonly humanperipheral blood, but B cells for transformation may also be derivedfrom other sources including, but not limited to, lymph nodes, tonsil,spleen, tumor tissue, and infected tissues; Tissues are generally madeinto single cell suspensions prior to EBV transformation. Additionally,steps may be taken to either physically remove or inactivate T cells(e.g., by treatment with cyclosporin A) in B cell-containing samples,because T cells from individuals seropositive for anti-EBV antibodiescan suppress B cell immortalization by EBV.

In general, the sample containing human B cells is innoculated with EBV,and cultured for 3-4 weeks. A typical source of EBV is the culturesupernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs ofEBV transformation can generally be seen towards the end of the 3-4 weekculture period. By phase-contrast microscopy, transformed cells mayappear large, clear, hairy and tend to aggregate in tight clusters ofcells. Initially, EBV lines are generally polyclonal. However, overprolonged periods of cell cultures, EBV lines may become monoclonal orpolyclonal as a result of the selective outgrowth of particular B cellclones. Alternatively, polyclonal EBV transformed lines may be subcloned(e.g., by limiting dilution culture) or fused with a suitable fusionpartner and plated at limiting dilution to obtain monoclonal B celllines. Suitable fusion partners for EBV transformed cell lines includemouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma celllines (human x mouse; e.g, SPAM-8, SBC-H₂O, and CB-F7), and human celllines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the presentinvention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragmentsthereof, comprising EBV-transformation of human B cells.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, antibodies that bind to a Therapeutic protein can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make antibodies that bind to a Therapeutic proteininclude those disclosed in Brinkman et al., J. Immunol. Methods182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994>; PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston-et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody and fragments thereof. The invention alsoencompasses polynucleotides that hybridize under stringent oralternatively, under lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a Therapeutic protein, preferably, anantibody that binds to a polypeptide having the amino acid sequence of a“Therapeutic Protein X” as discosed in the “Exemplary Identifier” columnof Table 1.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art(see, Example 60).

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454-(1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Recombinant Expression of Antibodies

Recombinant expression of an antibody, or fragment, derivative or analogthereof, (e.g., a heavy or light chain of an antibody or a single chainantibody), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody. Thus, the invention includes hostcells containing a polynucleotide encoding an antibody of the invention,or a heavy or light chain thereof, or a single chain antibody, operablylinked to a heterologous promoter. In preferred embodiments for theexpression of double-chained antibodies, vectors encoding both the heavyand light chains may be co-expressed in the host cell for expression ofthe entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro,which confers resistance to hygromycin (Santerre et al., Gene 30:147(1984)). Methods commonly known in the art of recombinant DNA technologymay be routinely applied to select the desired recombinant clone, andsuch methods are described, for example, in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);Kriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds),Current Protocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availabilty of cell lines (e.g., themurine myeloma cell line, NS0) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. A glutamine synthase expression system and componentsthereof are detailed in PCT publications: WO87/04462; WO86/05807;WO89/01036; WO89/10404; and WO91/06657 which are incorporated in theirentireties by reference herein. Additionally, glutamine synthaseexpression vectors that may be used according to the present inventionare commercially available from suppliers, including, for example LonzaBiologics, Inc. (Portsmouth, N.H.). Expression and production ofmonoclonal antibodies using a GS expression system in murine myelomacells is described in Bebbington et al., Bio/technology 10:169(1992) andin Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which areincorporated in their entirities by reference herein.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies that bind to aTherapeutic protein and that may correspond to a Therapeutic proteinportion of an albumin fusion protein of the invention or fragmentsthereof can be fused to heterologous polypeptide sequences describedherein or otherwise known in the art, to facilitate purification.

Modifications of Antibodies

Antibodies that bind a Therapeutic protein or fragments or variants canbe fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc. Other examples of detectablesubstances have been described elsewwhere herein.

Further, an antibody of the invention may be conjugated to a therapeuticmoiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters suchas, for example, 213Bi. A cytotoxin or cytotoxic agent includes anyagent that is detrimental to cells. Examples include paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Antibody-Albumin Fusion

Antibodies that bind to a Therapeutic protein and that may correspond toa Therapeutic protein portion of an albumin fusion protein of theinvention include, but are not limited to, antibodies that bind aTherapeutic protein disclosed in the “Therapeutic Protein X” column ofTable 1, or a fragment or variant thereof.

In specific embodiments, the fragment or variant of an antibody thatspecifically binds a Therapeutic protein and that corresponds to aTherapeutic protein portion of an albumin fusion protein comprises, oralternatively consists of, the VH domain. In other embodiments, thefragment or variant of an antibody that specifically binds a Therapeuticprotein and that corresponds to a Therapeutic protein portion of analbumin fusion protein comprises, or alternatively consists of, one, twoor three VH CDRs. In other embodiments, the fragment or variant of anantibody that specifically binds a Therapeutic protein and thatcorresponds to a Therapeutic protein portion of an albumin fusionprotein comprises, or alternatively consists of, the VH CDR1. In otherembodiments, the fragment or variant of an antibody that specificallybinds a Therapeutic protein and that corresponds to a Therapeuticprotein portion of an albumin fusion protein comprises, or alternativelyconsists of, the VH CDR2. In other embodiments, the fragment or variantof an antibody that specifically binds a Therapeutic protein and thatcorresponds to a Therapeutic protein portion of an albumin fusionprotein comprises, or alternatively consists of, the VH CDR3.

In specific embodiments, the fragment or variant of an antibody thatspecifically binds a Therapeutic protein and that corresponds to aTherapeutic protein portion of an albumin fusion protein comprises, oralternatively consists of, the VL domain. In other embodiments, thefragment or variant of an antibody that specifically binds a Therapeuticprotein and that corresponds to a Therapeutic protein portion of analbumin fusion protein comprises, or alternatively consists of, one, twoor three VL CDRs. In other embodiments, the fragment or variant of anantibody that specifically binds a Therapeutic protein and thatcorresponds to a Therapeutic protein portion of an albumin fusionprotein comprises, or alternatively consists of, the VL CDR1. In otherembodiments, the fragment or variant of an antibody that specificallybinds a Therapeutic protein and that corresponds to a Therapeuticprotein portion of an albumin fusion protein comprises, or alternativelyconsists of, the VL CDR2. In other embodiments, the fragment or variantof an antibody that specifically binds a Therapeutic protein and thatcorresponds to a Therapeutic protein portion of an albumin fusionprotein comprises, or alternatively consists of, the VL CDR3.

In other embodiments, the fragment or variant of an antibody thatspecifically binds a Therapeutic protein and that corresponds to aTherapeutic protein portion of an albumin fusion protein comprises, oralternatively consists of, one, two, three, four, five, or six VH and/orVL CDRs.

In preferred embodiments, the fragment or variant of an antibody thatspecifically binds a Therapeutic protein and that corresponds to aTherapeutic protein portion of an albumin fusion protein comprises, oralternatively consists of, an scFv comprising the VH domain of theTherapeutic antibody, linked to the VL domain of the therapeuticantibody by a peptide linker such as (Gly₄Ser)₃ (SEQ ID NO:36).

Immunophenotyping

The antibodies of the invention or albumin fusion proteins of theinvention comprising at least a fragment or variant of an antibody thatbinds a Therapeutic protein (or fragment or variant thereof) may beutilized for immunophenotyping of cell lines and biological samples.Therapeutic proteins of the present invention may be useful ascell-specific markers, or more specifically as cellular markers that aredifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies (or albuminfusion proteins comprsing at least a fragment or variant of an antibodythat binds a Therapeutic protein) directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies (or albumin fusion proteins comprising atleast a fragment or variant of an antibody that binds a Therapeuticprotein) to screen for cellular populations expressing the marker(s),and include magnetic separation using antibody-coated magnetic beads,“panning” with antibody attached to a solid matrix (i.e., plate), andflow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (NRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Characterizing Antibodies that Bind a Therapeutic Protein and AlbuminFusion Proteins Comprising a Fragment or Variant of an Antibody thatBinds a Therapeutic Protein

The antibodies of the invention or albumin fusion proteins of theinvention comprising at least a fragment or variant of an antibody thatbinds a Therapeutic protein (or fragment or variant thereof) may becharacterized in a variety of ways. In particular, Albumin fusionproteins of the invention comprising at least a fragment or variant ofan antibody that binds a Therapeutic protein may be assayed for theability to specifically bind to the same antigens specifically bound bythe antibody that binds a Therapeutic protein corresponding to theantibody that binds a Therapeutic protein portion of the albumin fusionprotein using techniques described herein or routinely modifyingtechniques known in the art.

Assays for the ability of the antibodies of the invention or albuminfusion proteins of the invention comprising at least a fragment orvariant of an antibody that binds a Therapeutic protein (or fragment orvariant thereof) to (specifically) bind a specific protein or epitopemay be performed in solution (e.g., Houghten, Bio/Techniques13:412-421(1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), onchips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S.Pat. No. 5,223,409), on spores (e.g., U.S. Pat. Nos. 5,571,698;5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl.Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith,Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirlaet al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J.Mol. Biol. 222:301-310 (1991)) (each of these references is incorporatedherein in its entirety by reference). The antibodies of the invention oralbumin fusion proteins of the invention comprising at least a fragmentor variant of an antibody that binds a Therapeutic protein (or fragmentor variant thereof) may also be assayed for their specificity andaffinity for a specific protein or epitope using or routinely modifyingtechniques described herein or otherwise known in the art.

The albumin fusion proteins of the invention comprising at least afragment or variant of an antibody that binds a Therapeutic protein maybe assayed for cross-reactivity with other antigens (e.g., moleculesthat have sequence/structure conservation with the molecule(s)specifically bound by the antibody that binds a Therapeutic protein (orfragment or variant thereof) corresponding to the Therapeutic proteinportion of the albumin fusion protein of the invention) by any methodknown in the art.

Immunoassays which can be used to analyze (immunospecific) binding andcross-reactivity include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, and protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety). Exemplary immunoassays are described briefly below(but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding an antibody of the invention or albumin fusion protein of theinvention comprising at least a fragment or variant of an antibody thatbinds a Therapeutic protein (or fragment or variant thereof) to the celllysate, incubating for a period of time (e.g., 1 to 4 hours) at 40degrees C., adding protein A and/or protein G sepharose beads (or beadscoated with an appropriate anti-iditoypic antibody or anti-albuminantibody in the case when an albumin fusion protein comprising at leasta fragment or variant of a Therapeutic antibody) to the cell lysate,incubating for about an hour or more at 40 degrees C., washing the beadsin lysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody or albumin fusion protein of the invention toimmunoprecipitate a particular antigen can be assessed by, e.g., westernblot analysis. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the binding of the antibodyor albumin fusion protein to an antigen and decrease the background(e.g., pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), applying the antibodyor albumin fusion protein of the invention (diluted in blocking buffer)to the membrane, washing the membrane in washing buffer, applying asecondary antibody (which recognizes the antibody or albumin fusionprotein, e.g., an anti-human serum albumin antibody) conjugated to anenzymatic substrate (e.g., horseradish peroxidase or alkalinephosphatase) or radioactive molecule (e.g., ³²P or ¹²⁵I) diluted inblocking buffer, washing the membrane in wash buffer, and detecting thepresence of the antigen. One of skill in the art would be knowledgeableas to the parameters that can be modified to increase the signaldetected and to reduce the background noise. For further discussionregarding western blot protocols see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, washing away antigen that did notbind the wells, adding the antibody or albumin fusion protein(comprising at least a fragment or variant of an antibody that binds aTherapeutic protein) of the invention conjugated to a detectablecompound such as an enzymatic substrate (e.g., horseradish peroxidase oralkaline phosphatase) to the wells and incubating for a period of time,washing away unbound or non-specifically bound albumin fusion proteins,and detecting the presence of the antibody or albumin fusion proteinsspecifically bound to the antigen coating the well. In ELISAs theantibody or albumin fusion protein does not have to be conjugated to adetectable compound; instead, a second antibody (which recognizes theantibody or albumin fusion protein, respectively) conjugated to adetectable compound may be added to the well. Further, instead ofcoating the well with the antigen, antibody or the albumin fusionprotein may be coated to the well. In this case, the detectable moleculecould be the antigen conjugated to a detectable compound such as anenzymatic substrate (e.g., horseradish peroxidase or alkalinephosphatase). One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected as wellas other variations of ELISAs known in the art. For further discussionregarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at11.2.1.

The binding affinity of an albumin fusion protein to a protein, antigen,or epitope and the off-rate of an antibody- or albumin fusionprotein-protein/antigen/epitope interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., ³H or ¹²⁵I) with the antibody or albumin fusion protein of theinvention in the presence of increasing amounts of unlabeled antigen,and the detection of the antibody bound to the labeled antigen. Theaffinity of the antibody or albumin fusion protein of the presentinvention for a specific protein, antigen, or epitope and the bindingoff-rates can be determined from the data by Scatchard plot analysis.Competition with a second protein that binds the same protein, antigenor epitope as the antibody or albumin fusion protein, can also bedetermined using radioimmunoassays. In this case, the protein, antigenor epitope is incubated with an antibody or albumin fusion protein ofthe present invention conjugated to a labeled compound (e.g., ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondprotein that binds the same protein, antigen, or epuitope as the albuminfusion protein of the invention.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibody or albumin fusion proteins ofthe invention to a protein, antigen or epitope. BIAcore kinetic analysiscomprises analyzing the binding and dissociation of antibodies, albuminfusion proteins, or specific polypeptides, antigens or epitopes fromchips with immobilized specific polypeptides, antigens or epitopes,antibodies or albumin fusion proteins, respectively, on their surface.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention or albuminfusion proteins of the invention comprising at least a fragment orvariant of an antibody that binds a Therapeutic protein to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein), nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein),albumin fusion proteins of the invention comprising at least a fragmentor variant of an antibody that binds a Therapeutic protein, and nucleicacids encoding such albumin fusion proteins. The antibodies of theinvention or albumin fusion proteins of the invention comprising atleast a fragment or variant of an antibody that binds a Therapeuticprotein can be used to treat, inhibit or prevent diseases, disorders orconditions associated with aberrant expression and/or activity of aTherapeutic protein, including, but not limited to, any one or more ofthe diseases, disorders, or conditions described herein. The treatmentand/or prevention of diseases, disorders, or conditions associated withaberrant expression and/or activity of a Therapeutic protein includes,but is not limited to, alleviating symptoms associated with thosediseases, disorders or conditions antibodies of the invention or albuminfusion proteins of the invention comprising at least a fragment orvariant of an antibody that binds a Therapeutic protein may be providedin pharmaceutically acceptable compositions as known in the art or asdescribed herein.

In a specific and preferred embodiment, the present invention isdirected to antibody-based therapies which involve administeringantibodies of the invention or albumin fusion proteins of the inventioncomprising at least a fragment or variant of an antibody that binds aTherapeutic protein to an animal, preferably a mammal, and mostpreferably a human, patient for treating one or more diseases,disorders, or conditions, including but not limited to: neuraldisorders, immune system disorders, muscular disorders, reproductivedisorders, gastrointestinal disorders, pulmonary disorders,cardiovascular disorders, renal disorders, proliferative disorders,and/or cancerous diseases and conditions., and/or as described elsewhereherein. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (e.g., antibodies directed tothe full length protein expressed on the cell surface of a mammaliancell; antibodies directed to an epitope of a Therapeutic protein andnucleic acids encoding antibodies of the invention (including fragments,analogs and derivatives thereof and anti-idiotypic antibodies asdescribed herein). The antibodies of the invention can be used to treat,inhibit or prevent diseases, disorders or conditions associated withaberrant expression and/or activity of a Therapeutic protein, including,but not limited to, any one or more of the diseases, disorders, orconditions described herein. The treatment and/or prevention ofdiseases, disorders, or conditions associated with aberrant expressionand/or activity of a Therapeutic protein includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention or albumin fusion proteins ofthe invention comprising at least a fragment or variant of an antibodythat binds a Therapeutic protein may be provided in pharmaceuticallyacceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the invention oralbumin fusion proteins of the invention comprising at least a fragmentor variant of an antibody that binds a Therapeutic protein may be usedtherapeutically includes binding Therapeutic proteins locally orsystemically in the body or by direct cytotoxicity of the antibody, e.g.as mediated by complement (CDC) or by effector cells (ADCC). Some ofthese approaches are described in more detail below. Armed with theteachings provided herein, one of ordinary skill in the art will knowhow to use the antibodies of the invention or albumin fusion proteins ofthe invention comprising at least a fragment or variant of an antibodythat binds a Therapeutic protein for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of the invention or albumin fusion proteins of theinvention comprising at least a fragment or variant of an antibody thatbinds a Therapeutic protein may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention or albumin fusion proteins of theinvention comprising at least a fragment or variant of an antibody thatbinds a Therapeutic protein may be administered alone or in combinationwith other types of treatments (e.g., radiation therapy, chemotherapy,hormonal therapy, immunotherapy and anti-tumor agents). Generally,administration of products of a species origin or species reactivity (inthe case of antibodies) that is the same species as that of the patientis preferred. Thus, in a preferred embodiment, human antibodies,fragments derivatives, analogs, or nucleic acids, are administered to ahuman patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against Therapeutic proteins, fragmentsor regions thereof, (or the albumin fusion protein correlate of such anantibody) for both immunoassays directed to and therapy of disordersrelated to polynucleotides or polypeptides, including fragments thereof,of the present invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude dissociation constants or Kd's less than 5×10⁻² M, 10⁻² M,5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M. More preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻⁵ M,10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M or 10⁻⁸ M. Even morepreferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰⁻¹² M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹⁰⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies that bind Therapeutic proteins or albumin fusion proteinscomprising at least a fragment or varaint of an antibody that binds aTherapeutic protein are administered to treat, inhibit or prevent adisease or disorder associated with aberrant expression and/or activityof a Therapeutic protein, by way of gene therapy. Gene therapy refers totherapy performed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded protein that mediates a therapeuticeffect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are described inmore detail elsewhere in this application.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody. Ina preferred embodiment, the compound is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side-effects). The subject is preferably an animal, includingbut not limited to animals such as cows, pigs, horses, chickens, cats,dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, e.g., the brain; thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E.W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a Therapeuticprotein can be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

Diagnosis and Imaging

Labeled antibodies and derivatives and analogs thereof that bind aTherapeutic protein (or fragment or variant thereof) (including albuminfusion proteins comprising at least a fragment or variant of an antibodythat binds a Therapeutic protein), can be used for diagnostic purposesto detect, diagnose, or monitor diseases, disorders, and/or conditionsassociated with the aberrant expression and/or activity of Therapeuticprotein. The invention provides for the detection of aberrant expressionof a Therapeutic protein, comprising (a) assaying the expression of theTherapeutic protein in cells or body fluid of an individual using one ormore antibodies specific to the polypeptide interest and (b) comparingthe level of gene expression with a standard gene expression level,whereby an increase or decrease in the assayed Therapeutic proteinexpression level compared to the standard expression level is indicativeof aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the Therapeutic protein incells or body fluid of an individual using one or more antibodiesspecific to the Therapeutic protein or albumin fusion proteinscomprising at least a fragment of variant of an antibody specific to aTherapeutic protein, and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed Therapeutic protein gene expression level compared to thestandard expression level is indicative of a particular disorder. Withrespect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention or albumin fusion proteins comprising atleast a fragment of variant of an antibody specific to a Therapeuticprotein can be used to assay protein levels in a biological sample usingclassical immunohistological methods known to those of skill in the art(e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanenet al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase;radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S),tritium (3H), indium (112In), and technetium (99Tc); luminescent labels,such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin.

One facet of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a Therapeutic proteinin an animal, preferably a mammal and most preferably a human. In oneembodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the Therapeutic protein isexpressed (and for unbound labeled molecule to be cleared to backgroundlevel); c) determining background level; and d) detecting the labeledmolecule in the subject, such that detection of labeled molecule abovethe background level indicates that the subject has a particular diseaseor disorder associated with aberrant expression of the Therapeuticprotein. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody,antibody fragment, or albumin fusion protein comprising at least afragement or variant of an antibody that binds a Therapeutic proteinwill then preferentially accumulate at the location of cells whichcontain the specific Therapeutic protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody, preferably apurified antibody, in one or more containers. In a specific embodiment,the kits of the present invention contain a substantially isolatedpolypeptide comprising an epitope which is specifically immunoreactivewith an antibody included in the kit. Preferably, the kits of thepresent invention further comprise a control antibody which does notreact with the polypeptide of interest. In another specific embodiment,the kits of the present invention contain a means for detecting thebinding of an antibody to a polypeptide of interest (e.g., the antibodymay be conjugated to a detectable substrate such as a fluorescentcompound, an enzymatic substrate, a radioactive compound or aluminescent compound, or a second antibody which recognizes the firstantibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Albumin Fusion Proteins

The present invention relates generally to albumin fusion proteins andmethods of treating, preventing, or ameliorating diseases or disorders.As used herein, “albumin fusion protein” refers to a protein formed bythe fusion of at least one molecule of albumin (or a fragment or variantthereof) to at least one molecule of a Therapeutic protein (or fragmentor variant thereof). An albumin fusion protein of the inventioncomprises at least a fragment or variant of a Therapeutic protein and atleast a fragment or variant of human serum albumin, which are associatedwith one another, preferably by genetic fusion (i.e., the albumin fusionprotein is generated by translation of a nucleic acid in which apolynucleotide encoding all or a portion of a Therapeutic protein isjoined in-frame with a polynucleotide encoding all or a portion ofalbumin) or chemical conjugation to one another. The Therapeutic proteinand albumin protein, once part of the albumin fusion protein, may bereferred to as a “portion”, “region” or “moiety” of the albumin fusionprotein.

In one embodiment, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a Therapeutic protein (e.g.,as described in Table 1) and a serum albumin protein. In otherembodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a biologically active and/ortherapeutically active fragment of a Therapeutic protein and a serumalbumin protein. In other embodiments, the invention provides an albuminfusion protein comprising, or alternatively consisting of, abiologically active and/or therapeutically active variant of aTherapeutic protein and a serum albumin protein. In preferredembodiments, the serum albumin protein component of the albumin fusionprotein is the mature portion of serum albumin.

In further embodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a Therapeutic protein, and abiologically active and/or therapeutically active fragment of serumalbumin. In further embodiments, the invention provides an albuminfusion protein comprising, or alternatively consisting of, a Therapeuticprotein and a biologically active and/or therapeutically active variantof serum albumin. In preferred embodiments, the Therapeutic proteinportion of the albumin fusion protein is the mature portion of theTherapeutic protein.

In further embodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a biologically active and/ortherapeutically active fragment or variant of a Therapeutic protein anda biologically active and/or therapeutically active fragment or variant,of serum albumin. In preferred embodiments, the invention provides analbumin fusion protein comprising, or alternatively consisting of, themature portion of a Therapeutic protein and the mature portion of serumalbumin.

Preferably, the albumin fusion protein comprises HA as the N-terminalportion, and a Therapeutic protein as the C-terminal portion.Alternatively, an albumin fusion protein comprising HA as the C-terminalportion, and a Therapeutic protein as the N-terminal portion may also beused.

In other embodiments, the albumin fusion protein has a Therapeuticprotein fused to both the N-terminus and the C-terminus of albumin. In apreferred embodiment, the Therapeutic proteins fused at the N- andC-termini are the same Therapeutic proteins. In a preferred embodiment,the Therapeutic proteins fused at the N- and C-termini are differentTherapeutic proteins. In another preferred embodiment, the Therapeuticproteins fused at the N- and C-termini are different Therapeuticproteins which may be used to treat or prevent the same disease,disorder, or condition (e.g. as listed in the “Preferred Indication Y”column of Table 1). In another preferred embodiment, the Therapeuticproteins fused at the N- and C-termini are different Therapeuticproteins which may be used to treat or prevent diseases or disorders(e.g. as listed in the “Preferred Indication Y” column of Table 1) whichare known in the art to commonly occur in patients simultaneously.

In addition to albumin fusion protein in which the albumin portion isfused N-terminal and/or C-terminal of the Therapeutic protein portion,albumin fusion proteins of the invention may also be produced byinserting the Therapeutic protein or peptide of interest (e.g., aTherapeutic protein X as diclosed in Table 1, or an antibody that bindsa Therapeutic protein or a fragment or variant thereof) into an internalregion of HA. For instance, within the protein sequence of the HAmolecule a number of loops or turns exist between the end and beginningof a-helices, which are stabilized by disulphide bonds (see FIGS. 9-11).The loops, as determined from the crystal structure of HA (FIG. 13) (PDBidentifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E to 1E7I and 1UOR) for the mostpart extend away from the body of the molecule. These loops are usefulfor the insertion, or internal fusion, of therapeutically activepeptides, particularly those requiring a secondary structure to befunctional, or Therapeutic proteins, to essentially generate an albuminmolecule with specific biological activity.

Loops in human albumin structure into which peptides or polypeptides maybe inserted to generate albumin fusion proteins of the inventioninclude: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176,His247-Glu252, Glu266-Glu277, Glu280-His288, Ala362-Glu368,Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys560-Thr566. In morepreferred embodiments, peptides or polypeptides are inserted into theVal54-Asn61, Gln 170-Ala176, and/or Lys560-Thr566 loops of mature humanalbumin (SEQ ID NO:18).

Peptides to be inserted may be derived from either phage display orsynthetic peptide libraries screened for specific biological activity orfrom the active portions of a molecule with the desired function.Additionally, random peptide libraries may be generated withinparticular loops or by insertions of randomized peptides into particularloops of the HA molecule and in which all possible combinations of aminoacids are represented.

Such library(s) could be generated on HA or domain fragments of HA byone of the following methods:

(a) randomized mutation of amino acids within one or more peptide loopsof HA or HA domain fragments. Either one, more or all the residueswithin a loop could be mutated in this manner (for example see FIG. 10a);

(b) replacement of, or insertion into one or more loops of HA or HAdomain fragments (i.e., internal fusion) of a randomized peptide(s) oflength X_(n) (where X is an amino acid and n is the number of residues(for example see FIG. 10 b);

(c) N-, C- or N- and C-terminal peptide/protein fusions in addition to(a) and/or (b).

The HA or HA domain fragment may also be made multifunctional bygrafting the peptides derived from different screens of different loopsagainst different targets into the same HA or HA domain fragment.

In preferred embodiments, peptides inserted into a loop of human serumalbumin are peptide fragments or peptide variants of the Therapeuticproteins disclosed in Table 1. More particulary, the inventionencompasses albumin fusion proteins which comprise peptide fragments orpeptide variants at least 7 at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least20, at least 25, at least 30, at least 35, or at least 40 amino acids inlength inserted into a loop of human serum albumin. The invention alsoencompasses albumin fusion proteins which comprise peptide fragments orpeptide variants at least 7 at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least20, at least 25, at least 30, at least 35, or at least 40 amino acidsfused to the N-terminus of human serum albumin. The invention alsoencompasses albumin fusion proteins which comprise peptide fragments orpeptide variants at least 7 at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least20, at least 25, at least 30, at least 35, or at least 40 amino acidsfused to the C-terminus of human serum albumin.

Generally, the albumin fusion proteins of the invention may have oneHA-derived region and one Therapeutic protein-derived region. Multipleregions of each protein, however, may be used to make an albumin fusionprotein of the invention. Similarly, more than one Therapeutic proteinmay be used to make an albumin fusion protein of the invention. Forinstance, a Therapeutic protein may be fused to both the N- andC-terminal ends of the HA. In such a configuration, the Therapeuticprotein portions may be the same or different Therapeutic proteinmolecules. The structure of bifunctional albumin fusion proteins may berepresented as: X-HA-Y or Y-HA-X.

For example, an anti-BLyS™ scFv-HA-IFNα-2b fusion may be prepared tomodulate the immune response to IFNα-2b by anti-BLyS™ scFv. Analternative is making a bi (or even multi) functional dose of HA-fusionse.g. HA-IFNα-2b fusion mixed with HA-anti-BLyS™ scFv fusion or otherHA-fusions in various ratio's depending on function, half-life etc.

Bi- or multi-functional albumin fusion proteins may also be prepared totarget the Therapeutic protein portion of a fusion to a target organ orcell type via protein or peptide at the opposite terminus of HA.

As an alternative to the fusion of known therapeutic molecules, thepeptides could be obtained-by screening libraries constructed as fusionsto the N-, C- or N- and C-termini of HA, or domain fragment of HA, oftypically 6, 8, 12, 20 or 25 or X_(n) (where X is an amino acid (aa) andn equals the number of residues) randomized amino acids, and in whichall possible combinations of amino acids were represented. A particularadvantage of this approach is that the peptides may be selected in situon the HA molecule and the properties of the peptide would therefore beas selected for rather than, potentially, modified as might be the casefor a peptide derived by any other method then being attached to HA.

Additionally, the albumin fusion proteins of the invention may include alinker peptide between the fused portions to provide greater physicalseparation between the moieties and thus maximize the accessibility ofthe Therapeutic protein portion, for instance, for binding to itscognate receptor. The linker peptide may consist of amino acids suchthat it is flexible or more rigid.

The linker sequence may be cleavable by a protease or chemically toyield the growth hormone related moiety. Preferably, the protease is onewhich is produced naturally by the host, for example the S. cerevisiaeprotease kex2 or equivalent proteases.

Therefore, as described above, the albumin fusion proteins of theinvention may have the following formula R1-L-R2; R2-L-R1; orR1-L-R2-L-R1, wherein R1 is at least one Therapeutic protein, peptide orpolypeptide sequence, and not necessarily the same Therapeutic protein,L is a linker and R2 is a serum albumin sequence.

In preferred embodiments, Albumin fusion proteins of the inventioncomprising a Therapeutic protein have extended shelf life compared tothe shelf life the same Therapeutic protein when not fused to albumin.Shelf-life typically refers to the time period over which thetherapeutic activity of a Therapeutic protein in solution or in someother storage formulation, is stable without undue loss of therapeuticactivity. Many of the Therapeutic proteins are highly labile in theirunfused state. As described below, the typical shelf-life of theseTherapeutic proteins is markedly prolonged upon incorporation into thealbumin fusion protein of the invention.

Albumin fusion proteins of the invention with “prolonged” or “extended”shelf-life exhibit greater therapeutic activity relative to a standardthat has been subjected to the same storage and handling conditions. Thestandard may be the unfused full-length Therapeutic protein. When theTherapeutic protein portion of the albumin fusion protein is an analog,a variant, or is otherwise altered or does not include the completesequence for that protein, the prolongation of therapeutic activity mayalternatively be compared to the unfused equivalent of that analog,variant, altered peptide or incomplete sequence. As an example, analbumin fusion protein of the invention may retain greater than about100% of the therapeutic activity, or greater than about 105%, 110%,120%, 130%, 150% or 200% of the therapeutic activity of a standard whensubjected to the same storage and handling conditions as the standardwhen compared at a given time point.

Shelf-life may also be assessed in terms of therapeutic activityremaining after storage, normalized to therapeutic activity when storagebegan. Albumin fusion proteins of the invention with prolonged orextended shelf-life as exhibited by prolonged or extended therapeuticactivity may retain greater than about 50% of the therapeutic activity,about 60%, 70%, 80%, or 90% or more of the therapeutic activity of theequivalent unfused Therapeutic protein when subjected to the sameconditions. For example, as discussed in Example 1, an albumin fusionprotein of the invention comprising hGH fused to the full length HAsequence may retain about 80% or more of its original activity insolution for periods of up to 5 weeks or more under various temperatureconditions.

Expression of Fusion Proteins

The albumin fusion proteins of the invention may be produced asrecombinant molecules by secretion from yeast, a microorganism such as abacterium, or a human or animal cell line. Preferably, the polypeptideis secreted from the host cells. We have found that, by fusing the hGHcoding sequence to the HA coding sequence, either to the 5′ end or 3′end, it is possible to secrete the albumin fusion protein from yeastwithout the requirement for a yeast-derived pro sequence. This wassurprising, as other workers have found that a yeast derived prosequence was needed for efficient secretion of hGH in yeast.

For example, Hiramatsu et al. (Appl Environ Microbiol 56:2125 (1990);Appl Environ Microbiol 57:2052 (1991)) found that the N-terminal portionof the pro sequence in the Mucor pusillus rennin pre-pro leader wasimportant. Other authors, using the MFα-1 signal, have always includedthe MFα-1 pro sequence when secreting hGH. The pro sequences werebelieved to assist in the folding of the hGH by acting as anintramolecular chaperone. The present invention shows that HA orfragments of HA can perform a similar function.

Hence, a particular embodiment of the invention comprises a DNAconstruct encoding a signal sequence effective for directing secretionin yeast, particularly a yeast-derived signal sequence (especially onewhich is homologous to the yeast host), and the fused molecule of thefirst aspect of the invention, there being no yeast-derived pro sequencebetween the signal and the mature polypeptide.

The Saccharomyces cerevisiae invertase signal is a preferred example ofa yeast-derived signal sequence.

Conjugates of the kind prepared by Poznansky et al., (FEBS Lett. 239:18(1988)), in which separately-prepared polypeptides are joined bychemical cross-linking, are not contemplated.

The present invention also includes a cell, preferably a yeast celltransformed to express an albumin fusion protein of the invention. Inaddition to the transformed host cells themselves, the present inventionalso contemplates a culture of those cells, preferably a monoclonal(clonally homogeneous) culture, or a culture derived from a monoclonalculture, in a nutrient medium. If the polypeptide is secreted, themedium will contain the polypeptide, with the cells, or without thecells if they have been filtered or centrifuged away. Many expressionsystems are known and may be used, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae, Kluyveromyces lactis and Pichia pastoris, filamentous fungi(for example Aspergillus), plant cells, animal cells and insect cells.

Preferred yeast strains to be used in the production of albumin fusionproteins are D88, DXY1 and BXP10. D88 [leu2-3, leu2-122, can1, pra1,ubc4] is a derivative of parent strain AH22his⁺ (also known as DB1; see,e.g., Sleep et al. Biotechnology 8:4246 (1990)). The strain contains aleu2 mutation which allows for auxotropic selection of 2 micron-basedplasmids that contain the LEU2 gene. D88 also exhibits a derepression ofPRB1 in glucose excess. The PRB1 promoter is normally controlled by twocheckpoints that monitor glucose levels and growth stage. The promoteris activated in wild type yeast upon glucose depletion and entry intostationary phase. Strain D88 exhibits the repression by glucose butmaintains the induction upon entry into stationary phase. The PRA1 geneencodes a yeast vacuolar protease, YscA endoprotease A, that islocalized in the ER. The UBC4 gene is in the ubiquitination pathway andis involved in targeting short lived and abnormal proteins for ubiquitindependant degradation. Isolation of this ubc4 mutation was found toincrease the copy number of an expression plasmid in the cell and causean increased level of expression of a desired protein expressed from theplasmid (see, e.g., International Publication No. WO99/00504, herebyincorporated in its entirety by reference herein).

DXY1, a derivative of D88, has the following genotype: [leu2-3,leu2-122, can1, pra1, ubc4, ura3::yap3]. In addition to the mutationsisolated in D88, this strain also has a knockout of the YAP3 protease.This protease causes cleavage of mostly di-basic residues (RR, RK, KR,KK) but can also promote cleavage at single basic residues in proteins.Isolation of this yap3 mutation resulted in higher levels of full lengthHSA production (see, e.g., U.S. Pat. No. 5,965,386 and Kerry-Williams etal., Yeast 14:161-169 (1998), hereby incorporated in their entireties byreference herein).

BXP10 has the following genotype: leu2-3, leu2-122, can1, pra1, ubc4,ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3. In addition to themutations isolated in DXY1, this strain also has a knockout of the PMT1gene and the HSP150 gene. The PMI1 gene is a member of theevolutionarily conserved family of dolichyl-phosphate-D-mannose proteinO-mannosyltransferases (Pmts). The transmembrane topology of Pmt1psuggests that it is an integral membrane protein of the endoplasmicreticulum with a role in O-linked glycosylation. This mutation serves toreduce/eliminate O-linked glycosylation of HSA fusions (see, e.g.,International Publication No. WO00/44772, hereby incorporated in itsentirety by reference herein. Studies revealed that the Hsp150 proteinis inefficiently separated from rHA by ion exchange chromatography. Themutation in the HSP150 gene removes a potential contaminant that hasproven difficult to remove by standard purification techniques. See,e.g., U.S. Pat. No. 5,783,423, hereby incorporated in its entirety byreference herein.

The desired protein is produced in conventional ways, for example from acoding sequence inserted in the host chromosome or on a free plasmid.The yeasts are transformed with a coding sequence for the desiredprotein in any of the usual ways, for example electroporation. Methodsfor transformation of yeast by electroporation are disclosed in Becker &Guarente (1990) Methods Enzymol. 194, 182.

Successfully transformed cells, i.e., cells that contain a DNA constructof the present invention, can be identified by well known techniques.For example, cells resulting from the introduction of an expressionconstruct can be grown to produce the desired polypeptide. Cells can beharvested and lysed and their DNA content examined for the presence ofthe DNA using a method such as that described by Southern (1975) J. Mol.Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208. Alternatively,the presence of the protein in the supernatant can be detected usingantibodies.

Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and aregenerally available from Stratagene Cloning Systems, La Jolla, Calif.92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markersHIS3, 7RP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (Ycps).

Preferred vectors for making albumin fusion proteins for expression inyeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA which aredescribed in detail in Example 2. FIG. 4 shows a map of the pPPC0005plasmid that can be used as the base vector into which polynucleotidesencoding Therapeutic proteins may be cloned to form HA-fusions. Itcontains a PRB1 S. cerevisiae promoter (PRB1p), a Fusion leader sequence(FL), DNA encoding HA (rHA) and an ADH1 S. cerevisiae terminatorsequence. The sequence of the fusion leader sequence consists of thefirst 19 amino acids of the signal peptide of human serum albumin (SEQID NO:29) and the last five amino acids of the mating factor alpha 1promoter (SLDKR, see EP-A-387 319 which is hereby incorporated byreference in its entirety.

The plasmids, pPPC0005, pScCHSA, pScNHSA, and pC4:HSA were deposited onApr. 11, 2001 at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 and given accession numbers ATCC______, ______, ______, and ______, respectively. Another vector usefulfor expressing an albumin fusion protein in yeast the pSAC35 vectorwhich is described in Sleep et al., BioTechnology 8:42 (1990) which ishereby incorporated by reference in its entirety.

A variety of methods have been developed to operably link DNA to vectorsvia complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion, is treatedwith bacteriophage T4 DNA polymerase or E. coli DNA polymerase I,enzymes that remove protruding, _-single-stranded termini with their 3′5′-exonucleolytic activities, and fill in recessed 3′-ends with theirpolymerizing activities.

The combination of these activities therefore generates blunt-ended DNAsegments. The blunt-ended segments are then incubated with a large molarexcess of linker molecules in the presence of an enzyme that is able tocatalyze the ligation of blunt-ended DNA molecules, such asbacteriophage T4 DNA ligase. Thus, the products of the reaction are DNAsegments carrying polymeric linker sequences at their ends. These DNAsegments are then cleaved with the appropriate restriction enzyme andligated to an expression vector that has been cleaved with an enzymethat produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., USA.

A desirable way to modify the DNA in accordance with the invention, if,for example, HA variants are to be prepared, is to use the polymerasechain reaction as disclosed by Saiki et al. (1988) Science 239, 487-491.In this method the DNA to be enzymatically amplified is flanked by twospecific oligonucleotide primers which themselves become incorporatedinto the amplified DNA. The specific primers may contain restrictionendonuclease recognition sites which can be used for cloning intoexpression vectors using methods known in the art.

Exemplary genera of yeast contemplated to be useful in the practice ofthe present invention as hosts for expressing the albumin fusionproteins are Pichia (formerly classified as Hansenula), Saccharomyces,Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora,Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces,Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora,Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,Sporidiobolus, Endomycopsis, and the like. Preferred genera are thoseselected from the group consisting of Saccharomyces,Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora. Examples ofSaccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.

Examples of Kluyveromyces spp. are K. fragilis, K. lactis and K.marxianus. A suitable Torulaspora species is T. delbrueckii. Examples ofPichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P.anomala (formerly H. anomala) and P. pastoris. Methods for thetransformation of S. cerevisiae are taught generally in EP 251 744, EP258 067 and WO 90/01063, all of which are incorporated herein byreference.

Preferred exemplary species of Saccharomyces include S. cerevisiae, S.italicus, S. diastaticus, and Zygosaccharomyces rouxii. Preferredexemplary species of Kluyveromyces include K. fragilis and K. lactis.Preferred exemplary species of Hansenula include H. polymorpha (nowPichia angusta), H. anomala (now Pichia anomala), and Pichia capsulata.Additional preferred exemplary species of Pichia include P. pastoris.Preferred exemplary species of Aspergillus include A. niger and A.nidulans. Preferred exemplary species of Yarrowia include Y. lipolytica.Many preferred yeast species are available from the ATCC. For example,the following preferred yeast species are available from the ATCC andare useful in the expression of albumin fusion proteins: Saccharomycescerevisiae Hansen, teleomorph strain BY4743 yap3 mutant (ATCC AccessionNo. 4022731); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743hsp150 mutant (ATCC Accession No. 4021266); Saccharomyces cerevisiaeHansen, teleomorph strain BY4743 pmt1 mutant (ATCC Accession No.4023792); Saccharomyces cerevisiae Hansen, teleomorph (ATCC AccessionNos. 20626; 44773; 44774; and 62995); Saccharomyces diastaticus Andrewset Gilliland ex van der Walt, teleomorph (ATCC Accession No. 62987);Kluyveromyces lactis(Dombrowski) van der Walt, teleomorph (ATCCAccession No. 76492); Pichia angusta(Teunisson et al.) Kurtzman,teleomorph deposited as Hansenula polymorpha de Morais et Maia,teleomorph (ATCC Accession No. 26012); Aspergillus niger van Tieghem,anamorph (ATCC Accession No. 9029); Aspergillus niger van Tieghem,anamorph (ATCC Accession No. 16404); Aspergillus nidulans (Eidam)Winter, anamorph (ATCC Accession No. 48756); and Yarrowia lipolytica(Wickerham et al.) van der Wait et von Arx, teleomorph (ATCC AccessionNo. 201847).

Suitable promoters for S. cerevisiae include those associated with thePGKI gene, GAL1 or GAL10 genes, CYCI, PHO5, TRPI, ADHI, ADH2, the genesfor glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, triose phosphate isomerase,phosphoglucose isomerase, glucokinase, alpha-mating factor pheromone, [amating factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDIpromoter, and hybrid promoters involving hybrids of parts of 5′regulatory regions with parts of 5′ regulatory regions of otherpromoters or with upstream activation sites (e.g. the promoter ofEP-A-258 067).

Convenient regulatable promoters for use in Schizosaccharomyces pombeare the thiamine-repressible promoter from the nmt gene as described byMaundrell (1990) J. Biol. Chem. 265, 10857-10864 and the glucoserepressible jbpl gene promoter as described by Hoffman & Winston (1990)Genetics 124, 807-816.

Methods of transforming Pichia for expression of foreign genes aretaught in, for example, Cregg et al. (1993), and various Phillipspatents (e.g. U.S. Pat. No. 4,857,467, incorporated herein byreference), and Pichia expression kits are commercially available fromInvitrogen BV, Leek, Netherlands, and Invitrogen Corp., San Diego,Calif. Suitable promoters include AOXI and AOX2. Gleeson et al. (1986)J. Gen. Microbiol. 132, 3459-3465 include information on Hansenulavectors and transformation, suitable promoters being MOX1 and FMD1;whilst EP 361 991, Fleer et al. (1991) and other publications fromRhone-Poulenc Rorer teach how to express foreign proteins inKluyveromyces spp., a suitable promoter being PGKI.

The transcription termination signal is preferably the 3′ flankingsequence of a eukaryotic gene which contains proper signals fortranscription termination and polyadenylation. Suitable 3′ flankingsequences may, for example, be those of the gene naturally linked to theexpression control sequence used, i.e. may correspond to the promoter.Alternatively, they may be different in which case the terminationsignal of the S. cerevisiae ADHI gene is preferred.

The desired albumin fusion protein may be initially expressed with asecretion leader sequence, which may be any leader effective in theyeast chosen. Leaders useful in S. cerevisiae include that from themating factor alpha polypeptide (MFα-1) and the hybrid leaders ofEP-A-387 319. Such leaders (or signals) are cleaved by the yeast beforethe mature albumin is released into the surrounding medium. Further suchleaders include those of S. cerevisiae invertase (SUC2) disclosed in JP62-096086 (granted as 911036516), acid phosphatase (PH05), thepre-sequence of MFα-1, 0 glucanase (BGL2) and killer toxin; S.diastaticus glucoarnylase Il; S. carlsbergensis α-galactosidase (MEL1);K. lactis killer toxin; and Candida glucoarnylase.

Additional Methods of Recombinant and Synthetic Production of AlbuminFusion Proteins

The present invention also relates to vectors containing apolynucleotide encoding an albumin fusion protein of the presentinvention, host cells, and the production of albumin fusion proteins bysynthetic and recombinant techniques. The vector may be, for example, aphage, plasmid, viral, or retroviral vector. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

The polynucleotides encoding albumin fusion proteins of the inventionmay be joined to a vector containing a selectable marker for propagationin a host. Generally, a plasmid vector is introduced in a precipitate,such as a calcium phosphate precipitate, or in a complex with a chargedlipid. If the vector is a virus, it may be packaged in vitro using anappropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418, glutamine synthase, or neomycin resistance for eukaryotic cellculture, and tetracycline, kanamycin or ampicillin resistance genes forculturing in E. coli and other bacteria. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells, suchas E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells,such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris(ATCC Accession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

In one embodiment, polynucleotides encoding an albumin fusion protein ofthe invention may be fused to signal sequences which will direct thelocalization of a protein of the invention to particular compartments ofa prokaryotic or eukaryotic cell and/or direct the secretion of aprotein of the invention from a prokaryotic or eukaryotic cell. Forexample, in E. coli, one may wish to direct the expression of theprotein to the periplasmic space. Examples of signal sequences orproteins (or fragments thereof) to which the albumin fusion proteins ofthe invention may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pelB signal sequence, the maltose binding protein (MBP)signal sequence, MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit, and thesignal sequence of alkaline phosphatase. Several vectors arecommercially available for the construction of fusion proteins whichwill direct the localization of a protein, such as the pMAL series ofvectors (particularly the pMAL-p series) available from New EnglandBiolabs. In a specific embodiment, polynucleotides albumin fusionproteins of the invention may be fused to the pelB pectate lyase signalsequence to increase the efficiency of expression and purification ofsuch polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos.5,576,195 and 5,846,818, the contents of which are herein incorporatedby reference in their entireties.

Examples of signal peptides that may be fused to an albumin fusionprotein of the invention in order to direct its secretion in mammaliancells include, but are not limited to, the MPIF-1 signal sequence (e.g.,amino acids 1-21 of GenBank Accession number AAB51134), thestanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:34), and aconsensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:35). Asuitable signal sequence that may be used in conjunction withbaculoviral expression systems is the gp67 signal sequence (e.g., aminoacids 1-19 of GenBank Accession Number AAA72759).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availabilty of cell lines (e.g., themurine myeloma cell line, NSO) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. A glutamine synthase expression system and componentsthereof are detailed in PCT publications: WO87/04462; WO86/05807;WO89/01036; WO89/10404; and

WO91/06657, which are hereby incorporated in their entireties byreference herein. Additionally, glutamine synthase expression vectorscan be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.).Expression and production of monoclonal antibodies using a GS expressionsystem in murine myeloma cells is described in Bebbington et al.,Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog.11:1 (1995) which are herein incorporated by reference.

The present invention also relates to host cells containing theabove-described vector constructs described herein, and additionallyencompasses host cells containing nucleotide sequences of the inventionthat are operably associated with one or more heterologous controlregions (e.g., promoter and/or enhancer) using techniques known of inthe art. The host cell can be a higher eukaryotic cell, such as amammalian cell (e.g., a human derived cell), or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. A host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., phosphorylation, cleavage) of proteins. Appropriatecell lines can be chosen to ensure the desired modifications andprocessing of the foreign protein expressed.

Introduction of the nucleic acids and nucleic acid constructs of theinvention into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. Such methods are described in many standard laboratorymanuals, such as Davis et al., Basic Methods In Molecular Biology(1986). It is specifically contemplated that the polypeptides of thepresent invention may in fact be expressed by a host cell lacking arecombinant vector.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., the coding sequence corresponding to aTherapeutic protein may be replaced with an albumin fusion proteincorresponding to the Therapeutic protein), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences such as forexample, an albumin fusion protein of the invention corresponding to theTherapeutic protein may be included). The genetic material operablyassociated with the endogenous polynucleotide may activate, alter,and/or amplify endogenous polynucleotides.

In addition, techniques known in the art may be used to operablyassociate heterologous polynucleotides (e.g., polynucleotides encodingan albumin protein, or a fragment or variant thereof) and/orheterologous control regions (e.g., promoter and/or enhancer) withendogenous polynucleotide sequences encoding a Therapeutic protein viahomologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issuedJun. 24, 1997; International Publication Number WO 96/29411;International Publication Number WO 94/12650; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature342:435-438 (1989), the disclosures of each of which are incorporated byreference in their entireties).

Albumin fusion proteins of the invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, hydrophobic charge interaction chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

In preferred embodiments the albumin fusion proteins of the inventionare purified using Anion Exchange Chromatography including, but notlimited to, chromatography on Q sepharose, DEAE sepharose, poros HQ,poros DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/SourceQ and DEAE, Fractogel Q and DEAE columns.

In specific embodiments the albumin fusion proteins of the invention arepurified using Cation Exchange Chromatography including, but not limitedto, SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP,Toyopearl CM, Resource/Source S and CM, Fractogel S and CM columns andtheir equivalents and comparables.

In specific embodiments the albumin fusion proteins of the invention arepurified using Hydrophobic Interaction Chromatography including, but notlimited to, Phenyl, Butyl, Methyl, Octyl, Hexyl-sepharose, poros Phenyl,Butyl, Methyl, Octyl, Hexyl, Toyopearl Phenyl, Butyl, Methyl, Octyl,Hexyl Resource/Source Phenyl, Butyl, Methyl, Octyl, Hexyl, FractogelPhenyl, Butyl, Methyl, Octyl, Hexyl columns and their equivalents andcomparables.

In specific embodiments the albumin fusion proteins of the invention arepurified using Size Exclusion Chromatography including, but not limitedto, sepharose S100, S200, S300, superdex resin columns and theirequivalents and comparables.

In specific embodiments the albumin fusion proteins of the invention arepurified using Affinity Chromatography including, but not limited to,Mimetic Dye affinity, peptide affinity and antibody affinity columnsthat are selective for either the HSA or the “fusion target” molecules.

In preferred embodiments albumin fusion proteins of the invention arepurified using one or more Chromatography methods listed above. In otherpreferred embodiments, albumin fusion proteins of the invention arepurified using one or more of the following Chromatography columns, Qsepharose FF column, SP Sepharose FF column, Q Sepharose HighPerformance Column, Blue Sepharose FF column, Blue Column, PhenylSepharose FF column, DEAE Sepharose FF, or Methyl Column.

Additionally, albumin fusion proteins of the invention may be purifiedusing the process described in PCT International Publication WO 00/44772which is herein incorporated by reference in its entirety. One of skillin the art could easily modify the process described therein for use inthe purification of albumin fusion proteins of the invention.

Albumin fusion proteins of the present invention may be recovered from:products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect, and mammaliancells. Depending upon the host employed in a recombinant productionprocedure, the polypeptides of the present invention may be glycosylatedor may be non-glycosylated. In addition, albumin fusion proteins of theinvention may also include an initial modified methionine residue, insome cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express albuminfusion proteins of the invention in a eukaryotic system. Pichia pastorisis a methylotrophic yeast which can metabolize methanol as its solecarbon source. A main step in the methanol metabolization pathway is theoxidation of methanol to formaldehyde using O₂. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O₂—Consequently, in a growth medium depending on methanol asa main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. SeeEllis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding an albumin fusion protein of the invention, as set forthherein, in a Pichea yeast system essentially as described in “PichiaProtocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg,eds. The Humana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a polypeptide of the invention by virtue ofthe strong AOX1 promoter linked to the Pichia pastoris alkalinephosphatase (PHO) secretory signal peptide (i.e., leader) locatedupstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a polynucleotide encoding an albuminfusion protein of the present invention, may be achieved by cloning theheterologous polynucleotide of the invention into an expression vectorsuch as, for example, pGAPZ or pGAPZalpha, and growing the yeast culturein the absence of methanol.

In addition, albumin fusion proteins of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co.,N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example,a polypeptide corresponding to a fragment of a polypeptide can besynthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the polypeptide sequence.Non-classical amino acids include, but are not limited to, to theD-isomers of the common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu,e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,fluoro-amino acids, designer amino acids such as b-methyl amino acids,Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

The invention encompasses albumin fusion proteins of the presentinvention which are differentially modified during or after translation,e.g., by glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand, etc.Any of numerous chemical modifications may be carried out by knowntechniques, including but not limited, to specific chemical cleavage bycyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄;acetylation, formylation, oxidation, reduction; metabolic synthesis inthe presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The albumin fusion proteins may also be modified with adetectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the protein.

Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include iodine (¹²¹I,¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium (⁹⁹Tc, ^(99m)Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹ Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶ Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.

In specific embodiments, albumin fusion proteins of the presentinvention or fragments or variants thereof are attached to macrocyclicchelators that associate with radiometal ions, including but not limitedto, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In a preferredembodiment, the radiometal ion associated with the macrocyclic chelatorsis ¹¹¹In. In another preferred embodiment, the radiometal ion associatedwith the macrocyclic chelator is ⁹⁰Y. In specific embodiments, themacrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, DOTA is attached to an antibody of theinvention or fragment thereof via linker molecule. Examples of linkermolecules useful for conjugating DOTA to a polypeptide are commonlyknown in the art—see, for example, DeNardo et al., Clin Cancer Res.4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7(1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); whichare hereby incorporated by reference in their entirety.

As mentioned, the albumin fusion proteins of the invention may bemodified by either natural processes, such as post-translationalprocessing, or by chemical modification techniques which are well knownin the art. It will be appreciated that the same type of modificationmay be present in the same or varying degrees at several sites in agiven polypeptide. Polypeptides of the invention may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclic polypeptidesmay result from posttranslation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992)).

Albumin fusion proteins of the invention and antibodies that bind aTherapeutic protein or fragments or variants thereof can be fused tomarker sequences, such as a peptide to facilitate purification. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

Further, an albumin fusion protein of the invention may be conjugated toa therapeutic moiety such as a cytotoxin, e.g., a cytostatic orcytocidal agent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon, B-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors. Techniques for conjugating suchtherapeutic moiety to proteins (e.g., albumin fusion proteins) are wellknown in the art.

Albumin fusion proteins may also be attached to solid supports, whichare particularly useful for immunoassays or purification of polypeptidesthat are bound by, that bind to, or associate with albumin fusionproteins of the invention. Such solid supports include, but are notlimited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

Albumin fusion proteins, with or without a therapeutic moiety conjugatedto it, administered alone or in combination with cytotoxic factor(s)and/or cytokine(s) can be used as a therapeutic.

In embodiments where the albumin fusion protein of the inventioncomprises only the VH domain of an antibody that binds a Therapeuticprotein, it may be necessary and/or desirable to coexpress the fusionprotein with the VL domain of the same antibody that binds a Therapeuticprotein, such that the VH-albumin fusion protein and VL protein willassociate (either covalently or non-covalently) post-translationally.

In embodiments where the albumin fusion protein of the inventioncomprises only the VL domain of an antibody that binds a Therapeuticprotein, it may be necessary and/or desirable to coexpress the fusionprotein with the VH domain of the same antibody that binds a Therapeuticprotein, such that the VL-albumin fusion protein and VH protein willassociate (either covalently or non-covalently) post-translationally.

Some Therapeutic antibodies are bispecific antibodies, meaning theantibody that binds a Therapeutic protein is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. In order to create an albumin fusion proteincorresponding to that Therapeutic protein, it is possible to create analbumin fusion protein which has an scFv fragment fused to both the N-and C-terminus of the albumin protein moiety. More particularly, thescFv fused to the N-terminus of albumin would correspond to one of theheavy/light (VH/VL) pairs of the original antibody that binds aTherapeutic protein and the scFv fused to the C-terminus of albuminwould correspond to the other heavy/light (VH/VL) pair of the originalantibody that binds a Therapeutic protein.

Also provided by the invention are chemically modified derivatives ofthe albumin fusion proteins of the invention which may provideadditional advantages such as increased solubility, stability andcirculating time of the polypeptide, or decreased immunogenicity (seeU.S. Pat. No. 4,179,337). The chemical moieties for derivitization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The albumin fusion proteins maybe modified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antiogenicity and other known effects of thepolyethylene glycol to a Therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000,70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, such as, for example, themethod disclosed in EP 0 401 384 (coupling PEG to G-CSF), hereinincorporated by reference; see also Malik et al., Exp. Hematol.20:1028-1035 (1992), reporting pegylation of GM-CSF using tresylchloride. For example, polyethylene glycol may be covalently boundthrough amino acid residues via reactive group, such as a free amino orcarboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the albumin fusion proteins of theinvention may be accomplished by any number of means. For example,polyethylene glycol may be attached to the albumin fusion protein eitherdirectly or by an intervening linker. Linkerless systems for attachingpolyethylene glycol to proteins are described in Delgado et al., Crit.Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern.J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No.5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each ofwhich are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (CISO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber of additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin International Publication No. WO 98/32466, the entire disclosure ofwhich is incorporated herein by reference. Pegylated protein productsproduced using the reaction chemistries set out herein are includedwithin the scope of the invention.

The number of polyethylene glycol moieties attached to each albuminfusion protein of the invention (i.e., the degree of substitution) mayalso vary. For example, the pegylated proteins of the invention may belinked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, ormore polyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The polypeptides of the invention can be recovered and purified fromchemical synthesis and recombinant cell cultures by standard methodswhich include, but are not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Well known techniques forrefolding protein may be employed to regenerate active conformation whenthe polypeptide is denatured during isolation and/or purification.

The presence and quantity of albumin fusion proteins of the inventionmay be determined using ELISA, a well known immunoassay known in theart. In one ELISA protocol that would be useful fordetecting/quantifying albumin fusion proteins of the invention,comprises the steps of coating an ELISA plate with an anti-human serumalbumin antibody, blocking the plate to prevent non-specific binding,washing the ELISA plate, adding a solution containing the albumin fusionprotein of the invention (at one or more different concentrations),adding a secondary anti-Therapeutic protein specific antibody coupled toa detectable label (as described herein or otherwise known in the art),and detecting the presence of the secondary antibody. In an alternateversion of this protocol, the ELISA plate might be coated with theanti-Therapeutic protein specific antibody and the labeled secondaryreagent might be the anti-human albumin specific antibody.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful to produce thealbumin fusion proteins of the invention. As described in more detailbelow, polynucleotides of the invention (encoding albumin fusionproteins) may be used in recombinant DNA methods useful in geneticengineering to make cells, cell lines, or tissues that express thealbumin fusion protein encoded by the polynucleotides encoding albuminfusion proteins of the invention.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. Additionalnon-limiting examples of gene therapy methods encompassed by the presentinvention are more thoroughly described elsewhere herein (see, e.g., thesections labeled “Gene Therapy”, and Examples 17 and 18).

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

Albumin fusion proteins of the invention are useful to provideimmunological probes for differential identification of the tissue(s)(e.g., immunohistochemistry assays such as, for example, ABCimmunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981))or cell type(s) (e.g., immunocytochemistry assays).

Albumin fusion proteins can be used to assay levels of polypeptides in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other methods useful for detecting protein gene expressioninclude immunoassays, such as the enzyme linked immunosorbent assay(ELISA) and the radioimmunoassay (RIA). Suitable assay labels are knownin the art and include enzyme labels, such as, glucose oxidase;radioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In),and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹ Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

Albumin fusion proteins of the invention can also be detected in vivo byimaging. Labels or markers for in vivo imaging of protein include thosedetectable by X-radiography, nuclear magnetic resonance (NMR) orelectron spin relaxtion (ESR). For X-radiography, suitable labelsinclude radioisotopes such as barium or cesium, which emit detectableradiation but are not overtly harmful to the subject. Suitable markersfor NMR and ESR include those with a detectable characteristic spin,such as deuterium, which may be incorporated into the albumin fusionprotein by labeling of nutrients given to a cell line expressing thealbumin fusion protein of the invention.

An albumin fusion protein which has been labeled with an appropriatedetectable imaging moiety, such as a radioisotope (for example, ¹³¹I,¹¹²In, ^(99m)Tc, (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), andtechnetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³ Xe), fluorine (¹⁸F,¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹ Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaque substance, or a materialdetectable by nuclear magnetic resonance, is introduced (for example,parenterally, subcutaneously or intraperitoneally) into the mammal to beexamined for immune system disorder. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of ^(99m)Tc. The labeled albumin fusion protein will thenpreferentially accumulate at locations in the body (e.g., organs, cells,extracellular spaces or matrices) where one or more receptors, ligandsor substrates (corresponding to that of the Therapeutic protein used tomake the albumin fusion protein of the invention) are located.Alternatively, in the case where the albumin fusion protein comprises atleast a fragment or variant of a Therapeutic antibody, the labeledalbumin fusion protein will then preferentially accumulate at thelocations in the body (e.g., organs, cells, extracellular spaces ormatrices) where the polypeptides/epitopes corresponding to those boundby the Therapeutic antibody (used to make the albumin fusion protein ofthe invention) are located. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982)). The protocols described therein could easily bemodified by one of skill in the art for use with the albumin fusionproteins of the invention.

In one embodiment, the invention provides a method for the specificdelivery of albumin fusion proteins of the invention to cells byadministering albumin fusion proteins of the invention (e.g.,polypeptides encoded by polynucleotides encoding albumin fusion proteinsof the invention and/or antibodies) that are associated withheterologous polypeptides or nucleic acids. In one example, theinvention provides a method for delivering a Therapeutic protein intothe targeted cell. In another example, the invention provides a methodfor delivering a single stranded nucleic acid (e.g., antisense orribozymes) or double stranded nucleic acid (e.g., DNA that can integrateinto the cell's genome or replicate episomally and that can betranscribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering albumin fusion proteins of the invention in associationwith toxins or cytotoxic prodrugs.

By “toxin” is meant one or more compounds that bind and activateendogenous cytotoxic effector systems, radioisotopes, holotoxins,modified toxins, catalytic subunits of toxins, or any molecules orenzymes not normally present in or on the surface of a cell that underdefined conditions cause the cell's death. Toxins that may be usedaccording to the methods of the invention include, but are not limitedto, radioisotopes known in the art, compounds such as, for example,antibodies (or complement fixing containing portions thereof) that bindan inherent or induced endogenous cytotoxic effector system, thymidinekinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonasexotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweedantiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includesa cytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or otherradioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co ⁶⁵Zn,⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn,⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium; luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin. In a specific embodiment, the invention providesa method for the specific destruction of cells (e.g., the destruction oftumor cells) by administering polypeptides of the invention orantibodies of the invention in association with the radioisotope ⁹⁰Y. Inanother specific embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention or antibodies of theinvention in association with the radioisotope ¹¹¹In. In a furtherspecific embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention or antibodies of theinvention in association with the radioisotope ¹³¹I.

Techniques known in the art may be applied to label polypeptides of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).

The albumin fusion proteins of the present invention are useful fordiagnosis, treatment, prevention and/or prognosis of various disordersin mammals, preferably humans. Such disorders include, but are notlimited to, those described herein under the section heading “BiologicalActivities,” below.

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression level of a certain polypeptide incells or body fluid of an individual using an albumin fusion protein ofthe invention; and (b) comparing the assayed polypeptide expressionlevel with a standard polypeptide expression level, whereby an increaseor decrease in the assayed polypeptide expression level compared to thestandard expression level is indicative of a disorder. With respect tocancer, the presence of a relatively high amount of transcript inbiopsied tissue from an individual may indicate a predisposition for thedevelopment of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the cancer.

Moreover, albumin fusion proteins of the present invention can be usedto treat or prevent diseases or conditions such as, for example, neuraldisorders, immune system disorders, muscular disorders, reproductivedisorders, gastrointestinal disorders, pulmonary disorders,cardiovascular disorders, renal disorders, proliferative disorders,and/or cancerous diseases and conditions. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor supressor), to activate the activity of a polypeptide (e.g., bybinding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

In particular, albumin fusion proteins comprising of at least a fragmentor variant of a Therapeutic antibody can also be used to treat disease(as described supra, and elsewhere herein). For example, administrationof an albumin fusion protein comprising of at least a fragment orvariant of a Therapeutic antibody can bind, and/or neutralize thepolypeptide to which the Therapeutic antibody used to make the albuminfusion protein immunospecifically binds, and/or reduce overproduction ofthe polypeptide to which the Therapeutic antibody used to make thealbumin fusion protein immunospecifically binds. Similarly,administration of an albumin fusion protein comprising of at least afragment or variant of a Therapeutic antibody can activate thepolypeptide to which the Therapeutic antibody used to make the albuminfusion protein immunospecifically binds, by binding to the polypeptidebound to a membrane (receptor).

At the very least, the albumin fusion proteins of the invention of thepresent invention can be used as molecular weight markers on SDS-PAGEgels or on molecular sieve gel filtration columns using methods wellknown to those of skill in the art. Albumin fusion proteins of theinvention can also be used to raise antibodies, which in turn may beused to measure protein expression of the Therapeutic protein, albuminprotein, and/or the albumin fusion protein of the invention from arecombinant cell, as a way of assessing transformation of the host cell,or in a biological sample. Moreover, the albumin fusion proteins of thepresent invention can be used to test the biological activitiesdescribed herein.

Diagnostic Assays

The compounds of the present invention are useful for diagnosis,treatment, prevention and/or prognosis of various disorders in mammals,preferably humans. Such disorders include, but are not limited to, thosedescribed for each Therapeutic protein in the corresponding row of Table1 and herein under the section headings “Immune Activity,” “BloodRelated Disorders,” “Hyperproliferative Disorders,” “Renal Disorders,”“Cardiovascular Disorders,” “Respiratory Disorders,” “Anti-AngiogenesisActivity,” “Diseases at the Cellular Level,” “Wound Healing andEpithelial Cell Proliferation,” “Neural Activity and NeurologicalDiseases,” “Endocrine Disorders,” “Reproductive System Disorders,”“Infectious Disease,” “Regeneration,” and/or “GastrointestinalDisorders,” infra.

For a number of disorders, substantially altered (increased ordecreased) levels of gene expression can be detected in tissues, cellsor bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid orspinal fluid) taken from an individual having such a disorder, relativeto a “standard” gene expression level, that is, the expression level intissues or bodily fluids from an individual not having the disorder.Thus, the invention provides a diagnostic method useful during diagnosisof a disorder, which involves measuring the expression level of the geneencoding a polypeptide in tissues, cells or body fluid from anindividual and comparing the measured gene expression level with astandard gene expression level, whereby an increase or decrease in thegene expression level(s) compared to the standard is indicative of adisorder. These diagnostic assays may be performed in vivo or in vitro,such as, for example, on blood samples, biopsy tissue or autopsy tissue.

The present invention is also useful as a prognostic indicator, wherebypatients exhibiting enhanced or depressed gene expression willexperience a worse clinical outcome By “assaying the expression level ofthe gene encoding a polypeptide” is intended qualitatively orquantitatively measuring or estimating the level of a particularpolypeptide (e.g. a polypeptide corresponding to a Therapeutic proteindisclosed in Table 1) or the level of the mRNA encoding the polypeptideof the invention in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level or mRNA level) orrelatively (e.g., by comparing to the polypeptide level or mRNA level ina second biological sample). Preferably, the polypeptide expressionlevel or mRNA level in the first biological sample is measured orestimated and compared to a standard polypeptide level or mRNA level,the standard being taken from a second biological sample obtained froman individual not having the disorder or being determined by averaginglevels from a population of individuals not having the disorder. As willbe appreciated in the art, once a standard polypeptide level or mRNAlevel is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source containingpolypeptides of the invention (including portions thereof) or mRNA. Asindicated, biological samples include body fluids (such as sera, plasma,urine, synovial fluid and spinal fluid) and tissue sources found toexpress the full length or fragments thereof of a polypeptide or mRNA.Methods for obtaining tissue biopsies and body fluids from mammals arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the polypeptides of the invention are then assayed usingany appropriate method. These include Northern blot analysis, S1nuclease mapping, the polymerase chain reaction (PCR), reversetranscription in combination with the polymerase chain reaction(RT-PCR), and reverse transcription in combination with the ligase chainreaction (RT-LCR).

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of polypeptidesthat bind to, are bound by, or associate with albumin fusion proteins ofthe invention, in a biological sample (e.g., cells and tissues),including determination of normal and abnormal levels of polypeptides.Thus, for instance, a diagnostic assay in accordance with the inventionfor detecting abnormal expression of polypeptides that bind to, arebound by, or associate with albumin fusion proteins compared to normalcontrol tissue samples may be used to detect the presence of tumors.Assay techniques that can be used to determine levels of a polypeptidethat bind to, are bound by, or associate with albumin fusion proteins ofthe present invention in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Assaying polypeptide levels in a biological sample can occur using anyart-known method.

Assaying polypeptide levels in a biological sample can occur using avariety of techniques. For example, polypeptide expression in tissuescan be studied with classical immunohistological methods (Jalkanen etal., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell.Biol. 105:3087-3096 (1987)). Other methods useful for detectingpolypeptide gene expression include immunoassays, such as the enzymelinked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).Suitable antibody assay labels are known in the art and include enzymelabels, such as, glucose oxidase, and radioisotopes, such as iodine(¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In),and technetium (^(99m)Tc), and fluorescent labels, such as fluoresceinand rhodamine, and biotin.

The tissue or cell type to be analyzed will generally include thosewhich are known, or suspected, to express the gene of interest (such as,for example, cancer). The protein isolation methods employed herein may,for example, be such as those described in Harlow and Lane (Harlow, E.and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety. The isolated cells canbe derived from cell culture or from a patient. The analysis of cellstaken from culture may be a necessary step in the assessment of cellsthat could be used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of thegene.

For example, albumin fusion proteins may be used to quantitatively orqualitatively detect the presence of polypeptides that bind to, arebound by, or associate with albumin fusion proteins of the presentinvention. This can be accomplished, for example, by immunofluorescencetechniques employing a fluorescently labeled albumin fusion proteincoupled with light microscopic, flow cytometric, or fluorimetricdetection.

In a preferred embodiment, albumin fusion proteins comprising at least afragment or variant of an antibody that immunospecifically binds atleast a Therapeutic protein disclosed herein (e.g., the Therapeuticproteins disclosed in Table 1) or otherwise known in the art may be usedto quantitatively or qualitatively detect the presence of gene productsor conserved variants or peptide fragments thereof. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody coupled with light microscopic, flowcytometric, or fluorimetric detection.

The albumin fusion proteins of the present invention may, additionally,be employed histologically, as in immunofluorescence, immunoelectronmicroscopy or non-immunological assays, for in situ detection ofpolypeptides that bind to, are bound by, or associate with an albuminfusion protein of the present invention. In situ detection may beaccomplished by removing a histological specimen from a patient, andapplying thereto a labeled antibody or polypeptide of the presentinvention. The albumin fusion proteins are preferably applied byoverlaying the labeled albumin fusion proteins onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the polypeptides that bind to, are bound by, orassociate with albumin fusion proteins, but also its distribution in theexamined tissue. Using the present invention, those of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Immunoassays and non-immunoassays that detect polypeptides that bind to,are bound by, or associate with albumin fusion proteins will typicallycomprise incubating a sample, such as a biological fluid, a tissueextract, freshly harvested cells, or lysates of cells which have beenincubated in cell culture, in the presence of a detectably labeledantibody capable of binding gene products or conserved variants orpeptide fragments thereof, and detecting the bound antibody by any of anumber of techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled albumin fusion proteinof the invention. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody or polypeptide.Optionally the antibody is subsequently labeled. The amount of boundlabel on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding a polypeptide (e.g., an albumin fusion protein, or polypeptidethat binds, is bound by, or associates with an albumin fusion protein ofthe invention.) Well-known supports or carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding to apolypeptide. Thus, the support configuration may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube, or theexternal surface of a rod. Alternatively, the surface may be flat suchas a sheet, test strip, etc. Preferred supports include polystyrenebeads. Those skilled in the art will know many other suitable carriersfor binding antibody or antigen, or will be able to ascertain the sameby use of routine experimentation.

The binding activity of a given lot of albumin fusion protein may bedetermined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

In addition to assaying polypeptide levels in a biological sampleobtained from an individual, polypeptide can also be detected in vivo byimaging. For example, in one embodiment of the invention, albumin fusionproteins of the invention are used to image diseased or neoplasticcells.

Labels or markers for in vivo imaging of albumin fusion proteins of theinvention include those detectable by X-radiography, NMR, MRI, CAT-scansor ESR. For X-radiography, suitable labels include radioisotopes such asbarium or cesium, which emit detectable radiation but are not overtlyharmful to the subject. Suitable markers for NMR and ESR include thosewith a detectable characteristic spin, such as deuterium, which may beincorporated into the albumin fusion protein by labeling of nutrients ofa cell line (or bacterial or yeast strain) engineered.

Additionally, albumin fusion proteins of the invention whose presencecan be detected, can be administered. For example, albumin fusionproteins of the invention labeled with a radio-opaque or otherappropriate compound can be administered and visualized in vivo, asdiscussed, above for labeled antibodies. Further, such polypeptides canbe utilized for in vitro diagnostic procedures.

A polypeptide-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, 131I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for a disorder. Itwill be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled albuminfusion protein will then preferentially accumulate at the locations inthe body which contain a polypeptide or other substance that binds to,is bound by or associates with an albumin fusion protein of the presentinvention. In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

One of the ways in which an albumin fusion protein of the presentinvention can be detectably labeled is by linking the same to a reporterenzyme and using the linked product in an enzyme immunoassay (EIA)(Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978,Diagnostic Horizons 2:1-7, Microbiological Associates QuarterlyPublication, Walkersville, Md.); Voller et al., J. Clin. Pathol.31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981);Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton,Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, KgakuShoin, Tokyo). The reporter enzyme which is bound to the antibody willreact with an appropriate substrate, preferably a chromogenic substrate,in such a manner as to produce a chemical moiety which can be detected,for example, by spectrophotometric, fluorimetric or by visual means.Reporter enzymes which can be used to detectably label the antibodyinclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for thereporter enzyme. Detection may also be accomplished by visual comparisonof the extent of enzymatic reaction of a substrate in comparison withsimilarly prepared standards.

Albumin fusion proteins may also be radiolabelled and used in any of avariety of other immunoassays. For example, by radioactively labelingthe albumin fusion proteins, it is possible to the use the albuminfusion proteins in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by means including, but not limited to, a gamma counter, ascintillation counter, or autoradiography.

It is also possible to label the albumin fusion proteins with afluorescent compound. When the fluorescently labeled antibody is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The albumin fusion protein can also be detectably labeled usingfluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the antibody using such metalchelating groups as diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

The albumin fusion proteins can also can be detectably labeled bycoupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged albumin fusion protein is then determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label albumin fusionproteins of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

Transgenic Organisms

Transgenic organisms that express the albumin fusion proteins of theinvention are also included in the invention. Transgenic organisms aregenetically modified organisms into which recombinant, exogenous orcloned genetic material has been transferred. Such genetic material isoften referred to as a transgene. The nucleic acid sequence of thetransgene may include one or more transcriptional regulatory sequencesand other nucleic acid sequences such as introns, that may be necessaryfor optimal expression and secretion of the encoded protein. Thetransgene may be designed to direct the expression of the encodedprotein in a manner that facilitates its recovery from the organism orfrom a product produced by the organism, e.g. from the milk, blood,urine, eggs, hair or seeds of the organism. The transgene may consist ofnucleic acid sequences derived from the genome of the same species or ofa different species than the species of the target animal. The transgenemay be integrated either at a locus of a genome where that particularnucleic acid sequence is not otherwise normally found or at the normallocus for the transgene.

The term “germ cell line transgenic organism” refers to a transgenicorganism in which the genetic alteration or genetic information wasintroduced into a germ line cell, thereby conferring the ability of thetransgenic organism to transfer the genetic information to offspring. Ifsuch offspring in fact possess some or all of that alteration or geneticinformation, then they too are transgenic organisms. The alteration orgenetic information may be foreign to the species of organism to whichthe recipient belongs, foreign only to the particular individualrecipient, or may be genetic information already possessed by therecipient. In the last case, the altered or introduced gene may beexpressed differently than the native gene.

A transgenic organism may be a transgenic animal or a transgenic plant.Transgenic animals can be produced by a variety of different methodsincluding transfection, electroporation, microinjection, gene targetingin embryonic stem cells and recombinant viral and retroviral infection(see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins etal. (1993) Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol.6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methodsin Molecular Biology No. 62, Humana Press (1997)). The method ofintroduction of nucleic acid fragments into recombination competentmammalian cells can be by any method which favors co-transformation ofmultiple nucleic acid molecules. Detailed procedures for producingtransgenic animals are readily available to one skilled in the art,including the disclosures in U.S. Pat. No. 5,489,743 and U.S. Pat. No.5,602,307.

A number of recombinant or transgenic mice have been produced, includingthose which express an activated oncogene sequence (U.S. Pat. No.4,736,866); express simian SV40 T-antigen (U.S. Pat. No. 5,728,915);lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Pat.No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No.5,723,719); express at least one human gene which participates in bloodpressure control (U.S. Pat. No. 5,731,489); display greater similarityto the conditions existing in naturally occurring Alzheimer's disease(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate cellularadhesion (U.S. Pat. No. 5,602,307); possess a bovine growth hormone gene(Clutter et al. (1996) Genetics 143(4):1753-1760); or, are capable ofgenerating a fully human antibody response (McCarthy (1997) The Lancet349(9049):405).

While mice and rats remain the animals of choice for most transgenicexperimentation, in some instances it is preferable or even necessary touse alternative animal species. Transgenic procedures have beensuccessfully utilized in a variety of non-murine animals, includingsheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits,cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev.46(4):515-526; Houdebine (1995) Reprod. Nutr. Dev. 35(6):609-617;Petters (1994) Reprod. Fertil. Dev. 6(5):643-645; Schnieke et al. (1997)Science 278(5346):2130-2133; and Amoah (1997) J. Animal Science75(2):578-585).

To direct the secretion of the transgene-encoded protein of theinvention into the milk of transgenic mammals, it may be put under thecontrol of a promoter that is preferentially activated in mammaryepithelial cells. Promoters that control the genes encoding milkproteins are preferred, for example the promoter for casein, betalactoglobulin, whey acid protein, or lactalbumin (see, e.g., DiTullio(1992) BioTechnology 10:74-77; Clark et al. (1989) BioTechnology7:487-492; Gorton et al. (1987) BioTechnology 5:1183-1187; and Soulieret al. (1992) FEBS Letts. 297:13). The transgenic mammals of choicewould produce large volumes of milk and have long lactating periods, forexample goats, cows, camels or sheep.

An albumin fusion protein of the invention can also be expressed in atransgenic plant, e.g. a plant in which the DNA transgene is insertedinto the nuclear or plastidic genome. Plant transformation proceduresused to introduce foreign nucleic acids into plant cells or protoplastsare known in the art (e.g., see Example 19). See, in general, Methods inEnzymology Vol. 153 (“Recombinant DNA Part D”) 1987, Wu and GrossmanEds., Academic Press and European Patent Application EP 693554. Methodsfor generation of genetically engineered plants are further described inU.S. Pat. No. 5,283,184, U.S. Pat. No. 5,482,852, and European PatentApplication EP 693 554, all of which are hereby incorporated byreference.

Pharmaceutical or Therapeutic Compositions

The albumin fusion proteins of the invention or formulations thereof maybe administered by any conventional method including parenteral (e.g.subcutaneous or intramuscular) injection or intravenous infusion. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time.

While it is possible for an albumin fusion protein of the invention tobe administered alone, it is preferable to present it as apharmaceutical formulation, together with one or more acceptablecarriers. The carrier(s) must be “acceptable” in the sense of beingcompatible with the albumin fusion protein and not deleterious to therecipients thereof. Typically, the carriers will be water or salinewhich will be sterile and pyrogen free. Albumin fusion proteins of theinvention are particularly well suited to formulation in aqueouscarriers such as sterile pyrogen free water, saline or other isotonicsolutions because of their extended shelf-life in solution. Forinstance, pharmaceutical compositions of the invention may be formulatedwell in advance in aqueous form, for instance, weeks or months or longertime periods before being dispensed.

For example, wherein the Therapeutic protein is hGH, EPO, alpha-IFN orbeta-IFN, formulations containing the albumin fusion protein may beprepared taking into account the extended shelf-life of the albuminfusion protein in aqueous formulations. As exhibited in Table 2, mostTherapeutic proteins are unstable with short shelf-lives afterformulation with an aqueous carrier. As discussed above, the shelf-lifeof many of these Therapeutic proteins are markedly increased orprolonged after fusion to HA. TABLE 2 Trade name, Storage ConditionsProtein Manufacturer Route Formulation of Non-Fusion Protein Interferon,alpha-2a Roferon-A, sc im sol_n (vial or pre-filled syringe) 4-8° C.Hoffmann-LaRoche Interferon, alpha-2b Intron-A, iv sc im sol_n; powder +dil. 4-8° C. Schering Plough (all preps, before and after dilution)COMBO Interferon Rebetron (Intron-A + po + sc Rebetol capsule + Intron-Ainjection alpha-2b + Ribavirin Rebetol) Schering Plough Interferon,Alphacon-1 Infergen Amgen sc sol_n 4-8° C. Interferon, alpha-nl,Wellferon, Wellcome sc im sol_n (with albumin _as stablizer_) 4-8° C.Lympho-blastoid Interferon, beta-1a Avonex, Biogen im powder + dil.(with albumin) 4-8° C. (before and after dilution) (Use within 3-6 h ofreconstitution) Rebif, Ares-Serono sc sol_n, in pre-filled syringe(Europe only) Interferon, beta-1b Betaseron, Chiron sc powder + dil.(with albumin) 4-8° C. (before and after dilution) (Europe: Betaferon)(Use within 3 h of reconstitution) Single use vials. Interferon,Gamma-1b Actimmune, InterMune sc 4-8° C. (before and after dilution)Pharmaceuticals (Use within 3 h of reconstitution). Growth HormoneGenotropin, powder/dil cartridges 4-8° C. (before and after dilution);(somatropin) Pharmacia Upjohn (single or multi-use); single useMiniQuick Delivery Device single use MiniQuick injector should berefrigerated until use. Humatrope, sc im powder + dil. 4-8° C. (beforeand after dilution) Eli Lilly (Vial or pen cartridge) (Use vials within25 h, cartridges within 28 d, of reconstitution). Norditropin, NovoNordisk Pharmaceuticals Nutropin, Genentech sc powder + dil. 4-8° C.(stable for 14 d after dil_n) (all preps, before and after dilution)Nutropin AQ, sc sol_n 4-8° C. (Stable for 28 d after 1st use) GenentechNutropin Depot, sc microsphere suspension as 4-8° C. Single use pkges.Dose Genentech powder + dil. 1-2×/month (ProLease micro- encapsulationtechnol.) Saizen, (Serono) sc im powder + dil. Powder _should be storedat Rm Temp_. After reconstitution store 4-8° C. for up to 14 d.Serostim, Serono Powder _should be stored at Rm Temp_. Afterreconstitution store in 4-8° C. for up to 14 d. hGH, with N-term. MetProtropin, sc im powder + dil. 4-8° C. (all preps, before and after(somatrem) Genentech dilution) Erythropoietin Epogen, Amgen iv sc sol_n4-8° C. (use within 21 d of first use) (Epoetin alfa) (Single &multi-dose vials) Procrit, Amgen iv sc sol_n 4-8° C. (use within 21 d offirst use) (Single & multi-dose vials)

In instances where aerosol administration is appropriate, the albuminfusion proteins of the invention can be formulated as aerosols usingstandard procedures. The term “aerosol” includes any gas-borne suspendedphase of an albumin fusion protein of the instant invention which iscapable of being inhaled into the bronchioles or nasal passages.Specifically, aerosol includes any gas-borne suspension of droplets ofan albumin fusion protein of the instant invention, as may be producedin a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosolalso includes a dry powder composition of a compound of the instantinvention suspended in air or other carrier gas, which may be deliveredby insufflation from an inhaler device, for example. See Ganderton &Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987);Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems6:273-313; and Raeburn et al,. (1992) Pharmacol. Toxicol. Methods27:143-159.

The formulations of the invention are also typically non-immunogenic, inpart, because of the use of the components of the albumin fusion proteinbeing derived from the proper species. For instance, for human use, boththe Therapeutic protein and albumin portions of the albumin fusionprotein will typically be human. In some cases, wherein either componentis non human-derived, that component may be humanized by substitution ofkey amino acids-so that specific epitopes appear to the human immunesystem to be human in nature rather than foreign.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the albuminfusion protein with the carrier that constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes' which render the formulationappropriate for the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampules, vials or syringes, and may bestored in a freeze-dried (lyophilised) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders. Dosageformulations may contain the Therapeutic protein portion at a lowermolar concentration or lower dosage compared to the non-fused standardformulation for the Therapeutic protein given the extended serumhalf-life exhibited by many of the albumin fusion proteins of theinvention.

As an example, when an albumin fusion protein of the invention comprisesgrowth hormone as one or more of the Therapeutic protein regions, thedosage form can be calculated on the basis of the potency of the albuminfusion protein relative to the potency of hGH, while taking into accountthe prolonged serum half-life and shelf-life of the albumin fusionproteins compared to that of native hGH. Growth hormone is typicallyadministered at 0.3 to 30.0 IU/kg/week, for example 0.9 to 12.0IU/kg/week, given in three or seven divided doses for a year or more. Inan albumin fusion protein consisting of full length HA fused to fulllength GH, an equivalent dose in terms of units would represent agreater weight of agent but the dosage frequency can be reduced, forexample to twice a week, once a week or less.

Formulations or compositions of the invention may be packaged togetherwith, or included in a kit with, instructions or a package insertreferring to the extended shelf-life of the albumin fusion proteincomponent. For instance, such instructions or package inserts mayaddress recommended storage conditions, such as time, temperature andlight, taking into account the extended or prolonged shelf-life of thealbumin fusion proteins of the invention. Such instructions or packageinserts may also address the particular advantages of the albumin fusionproteins of the inventions, such as the ease of storage for formulationsthat may require use in the field, outside of controlled hospital,clinic or office conditions. As described above, formulations of theinvention may be in aqueous form and may be stored under less than idealcircumstances without significant loss of therapeutic activity.

Albumin fusion proteins of the invention can also be included innutraceuticals. For instance, certain albumin fusion proteins of theinvention may be administered in natural products, including milk ormilk product obtained from a transgenic mammal which expresses albuminfusion protein. Such compositions can also include plant or plantproducts obtained from a transgenic plant which expresses the albuminfusion protein. The albumin fusion protein can also be provided inpowder or tablet form, with or without other known, additives, carriers,fillers and diluents. Nutraceuticals are described in Scott Hegenhart,Food Product Design, December 1993.

The invention also provides methods of treatment and/or prevention ofdiseases or disorders (such as, for example, any one or more of thediseases or disorders disclosed herein) by administration to a subjectof an effective amount of an albumin fusion protein of the invention ora polynucleotide encoding an albumin fusion protein of the invention(“albumin fusion polynucleotide”) in a pharmaceutically acceptablecarrier.

The albumin fusion protein and/or polynucleotide will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with the albumin fusion protein and/orpolynucleotide alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe albumin fusion protein administered parenterally per dose will be inthe range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the albumin fusion protein is typicallyadministered at a dose rate of about 1 ug/kg/hour to about 50ug/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

Albumin fusion proteins and/or polynucleotides can be are administeredorally, rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.“Pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any. The term “parenteral” as used hereinrefers to modes of administration which include intravenous;intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Albumin fusion proteins and/or polynucleotides of the invention are alsosuitably administered by sustained-release systems. Examples ofsustained-release albumin fusion proteins and/or polynucleotides areadministered orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,gels, drops or transdermal patch), bucally, or as an oral or nasalspray. “Pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion. Additional examples ofsustained-release albumin fusion proteins and/or polynucleotides includesuitable polymeric materials. (such as, for example, semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormirocapsules), suitable hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release albumin fusion proteins and/or polynucleotides alsoinclude liposomally entrapped albumin fusion proteins and/orpolynucleotides of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing thealbumin fusion protein and/or polynucleotide are prepared by methodsknown per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA)82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA)77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal Therapeutic.

In yet an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are delivered by way of a pump (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the albumin fusionprotein and/or polynucleotide is formulated generally by mixing it atthe desired degree of purity, in a unit dosage injectable form(solution, suspension, or emulsion), with a pharmaceutically acceptablecarrier, i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other compounds that are known to be deleterious tothe Therapeutic.

Generally, the formulations are prepared by contacting the albuminfusion protein and/or polynucleotide uniformly and intimately withliquid carriers or finely divided solid carriers or both. Then, ifnecessary, the product is shaped into the desired formulation.Preferably the carrier is a parenteral carrier, more preferably asolution that is isotonic with the blood of the recipient. Examples ofsuch carrier vehicles include water, saline, Ringer's solution, anddextrose solution. Non-aqueous vehicles such as fixed oils and ethyloleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyargrinine or tripeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The albumin fusion protein is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Albumin fusionproteins and/or polynucleotides generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

Albumin fusion proteins and/or polynucleotides ordinarily will be storedin unit or multi-dose containers, for example, sealed ampoules or vials,as an aqueous solution or as a lyophilized formulation forreconstitution. As an example of a lyophilized formulation, 10-ml vialsare filled with 5 ml of sterile-filtered 1% (w/v) aqueous albumin fusionprotein and/or polynucleotide solution, and the resulting mixture islyophilized. The infusion solution is prepared by reconstituting thelyophilized albumin fusion protein and/or polynucteotide usingbacteriostatic Water-for-Injection.

In a specific and preferred embodiment, the Albumin fusion proteinformulations comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride,0:16 micromole sodium octanoate/milligram of fusion protein, 0.15micrograms/milliliter polysorbate 80, pH 7.2. In another specific andpreferred embodiment the Albumin fusion protein formulations consists0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodiumoctanoate/milligram of fusion protein, 15 micrograms/milliliterpolysorbate 80, pH 7.2. The pH and buffer are chosen to matchphysiological conditions and the salt is added as a tonicifier. Sodiumoctanoate has been chosen due to its reported ability to increase thethermal stability of the protein in solution. Finally, polysorbate hasbeen added as a generic surfactant, which lowers the surface tension ofthe solution and lowers non-specific adsorption of the albumin fusionprotein to the container closure system.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thealbumin fusion proteins and/or polynucleotides of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the albumin fusion proteins and/or polynucleotides may beemployed in conjunction with other therapeutic compounds.

The albumin fusion proteins and/or polynucleotides of the invention maybe administered alone or in combination with adjuvants. Adjuvants thatmay be administered with the albumin fusion proteins and/orpolynucleotides of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable preparationsof Corynebacterium parvum. In a specific embodiment, albumin fusionproteins and/or polynucleotides of the invention are administered incombination with alum. In another specific embodiment, albumin fusionproteins and/or polynucleotides of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to, Monophosphoryl lipid immunomodulator,AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, andVirosomal adjuvant technology. Vaccines that may be administered withthe albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The albumin fusion proteins and/or polynucleotides of the invention maybe administered alone or in combination with other therapeutic agents.Albumin fusion protein and/or polynucleotide agents that may beadministered in combination with the albumin fusion proteins and/orpolynucleotides of the invention, include but not limited to,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, and/ortherapeutic treatments described below. Combinations may be administeredeither concomitantly, e.g., as an admixture, separately butsimultaneously or concurrently; or sequentially. This includespresentations in which the combined agents are administered together asa therapeutic mixture, and also procedures in which the combined agentsare administered separately but simultaneously, e.g., as throughseparate intravenous lines into the same individual. Administration “incombination” further includes the separate administration of one of thecompounds or agents given first, followed by the second.

In one embodiment, the albumin fusion proteins and/or polynucleotides ofthe invention are administered in combination with an anticoagulant.Anticoagulants that may be administered with the compositions of theinvention include, but are not limited to, heparin, low molecular weightheparin, warfarin sodium (e.g., COUMADIN®), dicumarol,4-hydroxycoumarin, anisindione (e.g., MIRADON™), acenocoumarol (e.g.,nicoumalone, SINTHROME™), indan-1,3-dione, phenprocoumon (e.g.,MARCUMAR™), ethyl biscoumacetate (e.g., TROMEXAN™), and aspirin. In aspecific embodiment, compositions of the invention are administered incombination with heparin and/or warfarin. In another specificembodiment, compositions of the invention are administered incombination with warfarin. In another specific embodiment, compositionsof the invention are administered in combination with warfarin andaspirin. In another specific embodiment, compositions of the inventionare administered in combination with heparin. In another specificembodiment, compositions of the invention are administered incombination with heparin and aspirin.

In another embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withthrombolytic drugs. Thrombolytic drugs that may be administered with thecompositions of the invention include, but are not limited to,plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g.,KABIKINASE™), antiresplace (e.g., EMIINASE™), tissue plasminogenactivator (t-PA, altevase, ACTFIVASE™), urokinase (e.g., ABBOKINASE™),sauruplase, (Prourokinase, single chain urokinase), and aminocaproicacid (e.g., AMICAR™). In a specific embodiment, compositions of theinvention are administered in combination with tissue plasminogenactivator and aspirin.

In another embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withantiplatelet druas. Antiplatelet drugs that may be administered with thecompositions of the invention include, but are not limited to, aspirin,dipyridarmole (e.g., PERSANTINE™), and ticlopidine (e.g., TICLID™).

In specific embodiments, the use of anti-coagulants, thrombolytic and/orantiplatelet drugs in combination with albumin fusion proteins and/orpolynucleotides of the invention is contemplated for the prevention,diagnosis, and/or treatment of thrombosis, arterial thrombosis, venousthrombosis, thromboembolism, pulmonary embolism, atherosclerosis,myocardial infarction, transient ischemic attack, unstable angina. Inspecific embodiments, the use of anticoagulants, thrombolytic drugsand/or antiplatelet drugs in combination with albumin fusion proteinsand/or polynucleotides of the invention is contemplated for theprevention of occulsion of saphenous grafts, for reducing the risk ofperiprocedural thrombosis as might accompany angioplasty procedures, forreducing the risk of stroke in patients with atrial fibrillationincluding nonrheumatic atrial fibrillation, for reducing the risk ofembolism associated with mechanical heart valves and or mitral valvesdisease. Other uses for the therapeutics of the invention, alone or incombination with antiplatelet, anticoagulant, and/or thrombolytic drugs,include, but are not limited to, the prevention of occlusions inextracorporeal devices (e.g., intravascular canulas, vascular accessshunts in hemodialysis patients, hemodialysis machines, andcardiopulmonary bypass machines).

In certain embodiments, albumin fusion proteins and/or polynucleotidesof the invention are administered in combination with antiretroviralagents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs),non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/orprotease inhibitors (PIs). NRTIs that may be administered in combinationwith the albumin fusion proteins and/or polynucleotides of theinvention, include, but are not limited to, RETROVIR™ (zidovudine/AZT),VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™(stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). NNRTIs that may be administered in combinationwith the albumin fusion proteins and/or polynucleotides of theinvention, include, but are not limited to, VIRAMIUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the albumin fusion proteinsand/or polynucleotides of the invention, include, but are not limitedto, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir),and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviralagents, nucleoside reverse transcriptase inhibitors, non-nucleosidereverse transcriptase inhibitors, and/or protease inhibitors may be usedin any combination with albumin fusion proteins and/or polynucleotidesof the invention to treat AIDS and/or to prevent or treat HIV infection.

Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosineNRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/FIC; structurallyrelated to lamivudine (3TC) but with 3- to 10-fold greater activity invitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related tolamivudine but retains activity against a substantial proportion oflamivudine-resistant isolates; Biochem Pharma); Adefovir (refusedapproval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON®(Adefovir Dipivoxil, the active prodrug of adefovir; its active form isPMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG(active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC,with activity against AZT/3TC-resistant virus); GW420867X (GlaxoWellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87(3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl(SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (see,International Publication No. WO 98/17281).

Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI ofthe HEPT class; Triangle/Abbott); CAPRKVIRINE™ (AG-1549/S-1153, a nextgeneration NNRTI with activity against viruses containing the K103Nmutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity thanits predecessor delavirdine and is active against K103N mutants;Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivativesof efavirenz, designed to be active against viruses with the K103Nmutation; DuPont); GW420867X (has 25-fold greater activity than HBY097and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A(naturally occurring agent from the latex tree; active against virusescontaining either or both the Y181C and K103N mutations); and Propolis(see, International Publication No. WO 99/49830).

Additional protease inhibitors include LOPINAVIR™ (ABT378/r; AbbottLaboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb);TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia &Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinaviranalog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776(a peptidomimetic with in vitro activity against proteaseinhibitor-resistant viruses; Agouron); VX-175/GW433908 (phosphateprodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); andAGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

Additional antiretroviral agents include fusion inhibitors/gp41 binders.Fusion inhibitors/gp41 binders include T-20 (a peptide from residues643-678 of the HIV gp41 transmembrane protein ectodomain which binds togp41 in its resting state and prevents transformation to the fusogenicstate; Trimeris) and T-1249 (a second-generation fusion inhibitor;Trimeris).

Additional antiretroviral agents include fusion inhibitors/chemokinereceptor antagonists. Fusion inhibitors/chemokine receptor antagonistsinclude CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and itsanalogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acidpeptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonistssuch as RANTES (9-63), AOP-RANTES, NNY-RANTES, and TAK-779; andCCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Alsoincluded are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetoragonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibitfusion.

Additional antiretroviral agents include integrase inhibitors. Integraseinhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (adicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and relatedanthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably actsat cell surface rather than being a true integrase inhibitor; Arondex);and naphthols such as those disclosed in WO 98/50347.

Additional antiretroviral agents include hydroxyurea-like compunds suchas BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst);ribonucleotide reductase inhibitors such as DIDOX™ (Molecules forHealth); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha asVX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolatemofetil; Roche).

Additional antiretroviral agents include inhibitors of viral integrase,inhibitors of viral genome nuclear translocation such as arylenebis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES,NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES andglycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc fingerinhibitors such as dithiane compounds; targets of HIV Tat and Rev; andpharmacoenhancers such as ABT-378.

Other antiretroviral therapies and adjunct therapies include cytokinesand lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™(aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13;interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF,and IL-10; agents that modulate immune activation such as cyclosporinand prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003(Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinantenvelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120,gp120/soluble CD4 complex, Delta JR-FL protein, branched syntheticpeptide derived from discontinuous gp120 C3/C4 domain, fusion-competentimmunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapiessuch as genetic suppressor elements (GSEs; WO 98/54366), and intrakines(generically modified CC chemokines targetted to the ER to block surfaceexpression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72(1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as theanti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9,PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4,the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d,447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α:antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptoragonists and antagonists such as TCDD, 3,3′,4,4′, 5-pentachlorobiphenyl,3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (see, InternationalPublication No. WO 98/30213); and antioxidants such asγ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).

In a further embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination with anantiviral agent. Antiviral agents that may be administered with thealbumin fusion proteins and/or polynucleotides of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In other embodiments, albumin fusion proteins and/or polynucleotides ofthe invention may be administered in combination with anti-opportunisticinfection agents. Anti-opportunistic agents that may be administered incombination with the albumin fusion proteins and/or polynucleotides ofthe invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, albumin fusion proteinsand/or polynucleotides of the invention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE”, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, albumin fusion proteins and/or polynucleotides of theinvention are used in any combination with ISONIAZID™, RIFAMPIN™,PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or preventan opportunistic Mycobacterium avium complex infection. In anotherspecific embodiment, albumin fusion proteins and/or polynucleotides ofthe invention are used in any combination with RIFABUTIN™,CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat orprevent an opportunistic Mycobacterium tuberculosis infection. Inanother specific embodiment, albumin fusion proteins and/orpolynucleotides of the invention are used in any combination withGANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat orprevent an opportunistic cytomegalovirus infection. In another specificembodiment, albumin fusion proteins and/or polynucleotides of theinvention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™,and/or KETOCONAZOLE™ to prophylactically treat or prevent anopportunistic fungal infection. In another specific embodiment, albuminfusion proteins and/or polynucleotides of the invention are used in anycombination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, albumin fusionproteins and/or polynucleotides of the invention are used in anycombination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylacticallytreat or prevent an opportunistic Toxoplasma gondii infection. Inanother specific embodiment, albumin fusion proteins and/orpolynucleotides of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination with anantibiotic agent. Antibiotic agents that may be administered with thealbumin fusion proteins and/or polynucleotides of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

In other embodiments, the albumin fusion proteins and/or polynucleotidesof the invention are administered in combination with immunestimulants.Immunostimulants that may be administered in combination with thealbumin fusion proteins and/or polynucleotides of the invention include,but are not limited to, levamisole (e.g., ERGAMISOL™), isoprinosine(e.g. INOSIPLEX™), interferons (e.g. interferon alpha), and interleukins(e.g., IL-2).

In other embodiments, albumin fusion proteins and/or polynucleotides ofthe invention are administered in combination with immunosuppressiveagents. Immunosuppressive agents that may be administered in combinationwith the albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells. Otherimmunosuppressive agents that may be administered in combination withthe albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to, prednisolone, methotrexate,thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine(BREDININ™), brequinar, deoxyspergualin, and azaspirane (SKF 105685),ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™, NEOPAL™, SANGDYA™(cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolatemotefil, of which the active metabolite is mycophenolic acid), IMURAN™(azathioprine), glucocorticosteroids, adrenocortical steroids, such asDELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ andMEXATE™ (methotrxate), OXSORALEN-ULTRTA™ (methoxsalen) and RAPAMUNE™(sirolimus). In a specific embodiment, immunosuppressants may be used toprevent rejection of organ or bone marrow transplantation.

In an additional embodiment, albumin fusion proteins and/orpolynucleotides of the invention are administered alone or incombination with one or more intravenous immune globulin preparations.Intravenous immune globulin preparations that may be administered withthe albumin fusion proteins and/or polynucleotides of the inventioninclude, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™,GAMMAGARD S/D™, ATGAM™ (antithymocyte glubulin), and GAMIMUNE™. In aspecific embodiment, albumin fusion proteins and/or polynucleotides ofthe invention are administered in combination with intravenous immuneglobulin preparations in transplantation “therapy (e.g., bone marrowtransplant).

In certain embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered alone or incombination with an anti-inflammatory agent. Anti-inflammatory agentsthat may be administered with the albumin fusion proteins and/orpolynucleotides of the invention include, but are not limited to,corticosteroids (e.g. betamethasone, budesonide, cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone,prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs.(e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate,mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin,phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, andtolmetin.), as well as antihistamines, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-angiogenic agent.Anti-angiogenic agents that may be administered with the compositions ofthe invention include, but are not limited to, Angiostatin (Entremed,Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.),anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel(Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, TissueInhibitor of Metalloproteinase-2, VEGI, Plasminogen ActivatorInhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of thelighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude, but are not limited to, platelet factor 4; protamine sulphate;sulphated chitin derivatives (prepared from queen crab shells), (Murataet al., Cancer Res. 51:22-26, (1991)); Sulphated PolysaccharidePeptidoglycan Complex (SP-PG) (the function of this compound may beenhanced by the presence of steroids such as estrogen, and tamoxifencitrate); Staurosporine; modulators of matrix metabolism, including forexample, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline,Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate;4propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al.,Biochem J. 236:475-480, (1992)); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,(1990)); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, (1987)); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;(Takeuchi et al., Agents Actions 36:312-3.16, (1992)); andmetalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within thecontext of the present invention include Thalidomide, (Celgene, Warren,N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkrnan J Pediatr.Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C.Storgard et al., J. Clin. Invest. 103:47-54 (1999));carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.);TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; PI-85; Prinomastat(AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and5-Fluorouracil.

Anti-angiogenic agents that may be administed in combination with thecompounds of the invention may work through a variety of mechanismsincluding, but not limited to, inhibiting proteolysis of theextracellular matrix, blocking the function of endothelialcell-extracellular matrix adhesion molecules, by antagonizing thefunction of angiogenesis inducers such as growth factors, and inhibitingintegrin receptors expressed on proliferating endothelial cells.Examples of anti-angiogenic inhibitors that interfere with extracellularmatrix proteolysis and which may be administered in combination with thecompositons of the invention include, but are not limited to, AG-3340(Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.),BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-2703-2A(Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford,UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenicinhibitors that act by blocking the function of endothelialcell-extracellular matrix adhesion molecules and which may beadministered in combination with the compositons of the inventioninclude, but are not lmited to, EMD-121974 (Merck KcgaA Darmstadt,Germany) and Vitaxin. (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg,Md.). Examples of anti-angiogenic agents that act by directlyantagonizing or inhibiting angiogenesis inducers and which may beadministered in combination with the compositons of the inventioninclude, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.),Anti-VEGF antibody (Genentech, S. San Francisco, Calif.),PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. SanFrancisco, Calif.), SU-5416 (Sugen/Pharmnacia Upjohn, Bridaewater,N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act toindirectly inhibit angiogenesis. Examples of indirect inhibitors ofangiogenesis which may be administered in combination with thecompositons of the invention include, but are not limited to, IM-862(Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nutley,N.J.), and Pentosan polysulfate (Georgetown University, Washington,D.C.).

In particular embodiments, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of an autoimmune disease,such as for example, an autoimmune disease described herein.

In a particular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of arthritis. In a moreparticular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of rheumatoid arthritis.

In another embodiment, the polynucleotides encoding a polypeptide of thepresent invention are administered in combination with an angiogenicprotein, or polynucleotides encoding an angiogenic protein. Examples ofangiogenic proteins that may be administered with the compositions ofthe invention include, but are not limited to acidic and basicfibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growthfactor alpha and beta, platelet-derived endothelial cell growth factor,platelet-derived crowth factor, tumor necrosis factor alpha, hepatocytegrowth factor, insulin-like growth factor, colony stimulating factor,macrophage colony stimulating factor, granulocyte/macrophage colonystimulating factor, and nitric oxide synthase.

In additional embodiments, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the albumin fusionproteins and/or polynucleotides of the invention include, but are notlimited to alkylating agents such as nitrogen mustards (for example,Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide,Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines andmethylmelamines (for example, Hexamethylmelamine and Thiotepa), alkylsulfonates (for example, Busulfan), nitrosoureas (for example,Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), andStreptozocin (streptozotocin)), triazenes (for example, Dacarbazine(DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs (forexample, Methotrexate (amethopterin)), pyrimidine analogs (for example,Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine;FudR), and Cytarabine (cytosine arabinoside)), purine analogs andrelated inhibitors (for example, Mercaptopurine (6-mercaptopurine;6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin(2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB,vinblastine sulfate)) and Vincristine (vincristine sulfate)),epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics(for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin;rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), andMitomycin (mitomycin C), enzymes (for example, L-Asparaginase),biological response modifiers (for example, Interferon-alpha andinterferon-alpha-2b), platinum coordination compounds (for example,Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone),substituted ureas (for example, Hydroxyurea), methylhydrazinederivatives (for example, Procarbazine (N-methylhydrazine; MIH),adrenocorticosteroids (for example, Prednisone), progestins (forexample, Hydroxyprogesterone caproate, Medroxyprogesterone,Medroxyproaesterone acetate, and Megestrol acetate), estrogens (forexample, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate,Estradiol, and Ethinyl estradiol), antiestrogens (for example,Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone),antiandrogens (for example, Flutamide), gonadotropin-releasing horomoneanalogs (for example, Leuprolide), other hormones and hormone analogs(for example, methyltestosterone, estramustine, estramustine phosphatesodium, chlorotrianisene, and testolactone), and others (for example,dicarbazine, glutamic acid, and mitotane).

In one embodiment, the compositions of the invention are administered incombination with one or more of the following drugs: infliximab (alsoknown as Remicade™ Centocor, Inc.), Trocade (Roche, RO-32-3555),Leflunomnide (also known as Arava™ from Hoechst Marion Roussel),Kineret™(an IL-1 Receptor antagonist also known as Anakinra from Amgen,Inc.)

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or combination of one or more of the components of CHOP.In one embodiment, the compositions of the invention are administered incombination with anti-CD20 antibodies, human monoclonal anti-CD20antibodies. In another embodiment, the compositions of the invention areadministered in combination with anti-CD20 antibodies and CHOP, oranti-CD20 antibodies and any combination of one or more of thecomponents of CHOP, particularly cyclophosphamide and/or prednisone. Ina specific embodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituximab and CHOP, or Rituximab and anycombination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. In a specific embodiment,compositions of the invention are administered in combination withtositumomab. In a further embodiment, compositions of the invention areadministered with tositumomab and CHOP, or tositumomab and anycombination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. The anti-CD20 antibodies mayoptionally be associated with radioisotopes, toxins or cytotoxicprodrugs.

In another specific embodiment, the compositions of the invention areadministered in combination Zevalin™. In a further embodiment,compositions of the invention are administered with Zevalin™ and CHOP,or Zevalin™ and any combination of one or more of the components ofCHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may beassociated with one or more radisotopes. Particularly preferred isotopesare ⁹⁰Y and ¹¹¹In.

In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withcytokines. Cytokines that may be administered with the albumin fusionproteins and/or polynucleotides of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,albumin fusion proteins and/or polynucleotides of the invention may beadministered with any interleukin, including, but not limited to,IL-1alpha, IL-1beta, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-13, IL-19,IL-20, and IL-21.

In one embodiment, the albumin fusion proteins and/or polynucleotides ofthe invention are administered in combination with members of the TNFfamily. TNF, TNF-related or TNF-like molecules that may be administeredwith the albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TRANK, TR9 (International Publication No. WO 98/56892), TR10(International Publication No. WO 98/54202), 312C2 (InternationalPublication No. WO 98/06842), and TR12, and soluble forms CD154, CD70,and CD153.

In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withangiogenic proteins. Angiogenic proteins that may be administered withthe albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PIGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth. Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in Germnan Patent Number DE19639601. The above mentionedreferences are herein incorporated by reference in their entireties.

In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withFibroblast Growth Factors. Fibroblast Growth Factors that may beadministered with the albumin fusion proteins and/or polynucleotides ofthe invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withhematopoietic growth factors. Hematopoietic growth factors that may beadministered with the albumin fusion proteins and/or polynucleotides ofthe invention include, but are not limited to, granulocyte macrophagecolony stimulating factor (GM-CSF) (sargramostim, LEUKINE™, PROKINE™),granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN™),macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin(epoetin alfa, EPOGEN™, PROCRIT™.), stem cell factor (SCF, c-kit ligand,steel factor), megakaryocyte colony stimulating factor, PIXY321 (aGMCSF/IL-3 fusion protein), interleukins, especially any one or more ofIL-1 through IL-12, interferon-gamma, or thrombopoietin.

In certain embodiments, albumin fusion proteins and/or polynucleotidesof the present invention are administered in combination with adrenergicblockers, such as, for example, acebutolol, atenolol, betaxolol,bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol,penbutolol, pindolol, propranolol, sotalol, and timolol.

In another embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination with anantiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis,digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide,lidocaine, mexiletine, moricizine, phenyloin, procainamide, N-acetylprocainamide, propafenonel propranolol, quinidine, sotalol, tocainide,and verapamil).

In another embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withdiuretic agents, such as carbonic anhydrase-inhibiting agents (e.g.,acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics(e.g., glycerin, isosorbide, mannitol, and urea), diuretics that inhibitNa⁺-K⁺-2Cl⁻ symport (e.g., furosemide, bumetanide, azosemide,piretanide, tripamide, ethacrynic acid, muzolimine; and torsemide),thiazide and thiazide-like diuretics (e.g., bendroflumethiazide,benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide,methyclothiazide, polythiazide, trichormethiazide, chlorthalidone,indapamide, metolazone, and quinethazone), potassium sparing diuretics(e.g., amiloride and triamterene), and mineralcorticoid receptorantagonists (e.g., spironolactone, canrenone, and potassium canrenoate).

In one embodiment, the albumin fusion proteins and/or polynucleotides ofthe invention are administered in combination with treatments forendocrine and/or hormone imbalance disorders. Treatments for endocrineand/or hormone imbalance disorders include, but are not limited to,¹²⁷I, radioactive isotopes of iodine such as ¹³¹I and ¹²³I; recombinantgrowth hormone, such as HUMATROPE™ (recombinant somatropin); growthhormone analogs such as PROTROPIN™ (somatrem); dopamine agonists such asPARLODEL™ (bromocriptine); somatostatin analogs such as SANDOSTATIN™(octreotide); gonadotropin preparations such as PREGNYL™, A.P.L.™ andPROFASI™ (chorionic gonadotropin (CG)), PERGONAL™ (menotropins), andMETRODIN™ (urofollitropin (uFSH)); synthetic human gonadotropinreleasing hormone preparations such as FACTREL™ and LUTREPULSE™(gonadorelin hydrochloride); synthetic gonadotropin agonists such asLUPRON™ (leuprolide acetate), SUPPRELIN™ (histrelin acetate), SYNAREL™(nafarelin acetate), and ZOLADEX™ (goserelin acetate); syntheticpreparations of thyrotropin-releasincg hormone such as RELEFACT TRH™ andTHYPINONE™ (protirelin); recombinant human TSH such as THYROGEN™;synthetic preparations of the sodium salts of the natural isomers ofthyroid hormones such as L-T₄™, SYNTHROID™ and LEVCTHROID™(levothyroxine sodium), L-T₃™, CTOMEL™ and TRIOSTAT™ (liothyroinesodium), and THYROLAR™ (liotrix); antithyroid compounds such as6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazoleand TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole);beta-adrenergic receptor antagonists such as propranolol and esmolol;Ca² channel blockers; dexamethasone and iodinated radiological contrastagents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodiumipodate).

Additional treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to, estrogens or congugated estrogens suchas ESTRACE™ (estradiol), ESTINYL™ (ethinyl estradiol), PREMARIN™,ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone), ESTROVIS™(quinestrol), ESTRADER™ (estradiol), DELESTROGEN™ and VALERGEN™(estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECT LA™(estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen),SEROPHENE™ and CLOVUD™ (clomiphene); progestins such as DURALUTIN™(hydroxyprogesterone caproate), MPA™ and DEPO-PROVERA™(medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™(megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ andAYGESTIN™ (norethindrone acetate); progesterone implants such asNORPLANT SYSTEM.™ (subdermal implants of norgestrel); antiprogestinssuch as RU 486™ (mifepristone); hormonal contraceptives such as ENOVID™(norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device thatreleases progesterone), LOESTRIN™, BREVICON™, MLODICON™, GENORA™,NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinylestradiol/norethindrone), LEVLEN™, NORDETTE™, TRI-LEVLEN™ andTRIPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™(ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodioldiacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™(norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinylestradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinylestradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), andOVRETTE™ (norgestrel).

Additional treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to, testosterone esters such as methenoloneacetate and testosterone undecanoate; parenteral and oral androgens suchas TESTOJECT-50™ (testosterone), TESTEX™ (testosterone propionate),DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™ (testosteronecypionate), DANOCRINE™ (danazol), HALOTESTIN™ (fluoxymesterone), ORETONMETHYL™, TESTRED™ and VIRLON™ (methyltestosterone), and OXANDRIN™(oxandrolone); testosterone transdermal systems such as TESTODERM™;androgen receptor antagonist and 5-alpha-reductase inhibitors such asANDROCUR™ (cyproterone acetate), EULEXIN™ (flutamide), and PROSCAR™(finasteride); adrenocorticotropic hormone preparations such asCORTROSYN™ (cosyntropin); adrenocortical steroids and their syntheticanalog-s such as ACLOVATE™ (alclometasone dipropionate), CYCLOCORT™(amcinonide), BECLOVENT™ and VANCERIL” (beclomethasone dipropionate),CELESTONE™ (betamethasone), BENISONE™ and UTICORT™ (betamethasonebenzoate), DIPROSONE™ (betamethasone dipropionate), CELESTONE PHOSPKATE™(betamethasone sodium phosphate), CELESTONE SOLUSPAN™ (betamethasonesodium phosphate and acetate), BETA-VAL™ and VALISONE-™ (betamethasonevalerate), TEMOVATE™ (clobetasol propionate), CLODERM™ (clocortolonepivalate), CORTEF™ and HYDROCORTONE™ (cortisol (hydrocortisone)),HYDROCORTONE ACETATE™ (cortisol (hydrocortisone) acetate), LOCOID™(cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE™ (cortisol(hydrocortisone) sodium phosphate), A-HYDROCORT™ and SOLU CORTEF™(cortisol (hydrocortisone) sodium succinate), WESTCORT™ (cortisol(hydrocortisone) valerate), CORTISONE ACETATE™ (cortisone acetate),DESOWEN™ and TRIDESILON™ (desonide), TOPICORT™ (desoximetasone),DECADRON™ (dexamethasone), DECADRON LA™ (dexamethasone acetate),DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™ (dexamethasone sodiumphosphate), FLORONE™ and MAXIFLOR™ (diflorasone diacetate), FLORINEFACETATE™ (fludrocortisone acetate), AEROBID™ and NASALIDE™(flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide), LIDEX™(fluocinonide), FLUOR-OP™ and FML™ (fluorometholone), CORDRAN™(flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone),MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™(methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™(methylprednisolone sodium succinate), ELOCON™ (mometasone furoate),HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone),ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodiumphosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™(prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™(triamcinolone acetonide), ARISTOCORT™ and KENACORT DIACETATE™(triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide);inhibitors of biosynthesis and action of adrenocortical steroids such asCYTADREN™ (aminoglutethimide), NIZORAL™ (ketoconazole), MODRASTANE™(trilostane), and METOPIRONE™ (metyrapone); bovine, porcine or humaninsulin or mixtures thereof; insulin analogs; recombinant human insulinsuch as HUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™and ORNASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ andTOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide,MICRONASE™, DIBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide),and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), ciglitazone,pioglitazone, and alpha-glucosidase inhibitors; bovine or porcineglucagon; somatostatins such as SANDOSTATIN™ (octreotide); anddiazoxides such as PROGLYCEM™ (diazoxide).

In one embodiment, the albumin fusion proteins and/or polynucleotides ofthe invention are administered in combination with treatments foruterine motility disorders. Treatments for uterine motility disordersinclude, but are not limited to, estrogen drugs such as conjugatedestrogens (e.g., PREMARIN® and ESTRATAB®), estradiols (e.g., CLIMARA®and ALORA®), estropipate, and chlorotrianisene; progestin drugs (e.g.,AMEN® (medroxyprogesterone), MICRONOR® (norethidrone acetate),PROETMRIUM® progesterone, and megestrol acetate); andestrogen/progesterone combination therapies such as, for example,conjugated estrogens/medroxyprogesterone (e.g., PREMPRO™ andPREMIPHASE®) and norethindrone acetate/ethinyl estsradiol (e.g.,FEMHRT®).

In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withdrugs effective in treating iron deficiency and hypochromic anemias,including but not limited to, ferrous sulfate (iron sulfate, FEOSOL™),ferrous fumarate (e.g., FEOSTAT™), ferrous gluconate (e.g. FERGON™)polysaccharide-iron complex (e.g., NIFEREX™), iron dextran injection(e.g., INFED™), cupric sulfate, pyroxidine, riboflavin, Vitamin B₁₂,cyancobalamin injection (e.g. REDISOL™, RUBRAMIN PC™), hydroxocobalamin,folic acid (e.g., FOLVITE™), leucovorin (folinic acid, 5-CHOH4PteGlu,citrovorum factor) or WELLCOVORIN (Calcium salt of leucovorin),transferrin or ferritin.

In certain embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withagents used to treat psychiatric disorders. Psychiatric drugs that maybe administered with the albumin fusion proteins and/or polynucleotidesof the invention include, but are not limited to, antipsychotic agents(e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine,haloperidol, loxapine, mesoridazine, molindone, olanzapine,perphenazine, pimozide, quetiapine, risperidone, thioridazine,thiothixene, trifluoperazine, and triflupromazine), antimanic agents(e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithiumcitrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion,citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine,imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone,nortriptyline, paroxetine, phenelzine, protriptyline, sertraline,tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxietyagents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate,diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants(e.g., d-amphetamine, methylphenidate, and pemoline).

In other embodiments, the albumin fusion proteins and/or polynucleotidesof the invention are administered in combination with agents used totreat neurological disorders. Neurological agents that may beadministered with the albumin fusion proteins and/or polynucleotides ofthe invention include, but are not limited to, antiepileptic agents(e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital,phenyloin, primidone, valproic acid, divalproex sodium, felbamate,gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine,topiramate, zonisamide, diazepam, lorazepam, and clonazepam),antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline,amantidine, bromocriptine, pergolide, ropinirole, pramipexole,benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl,tolcapone), and ALS therapeutics (e.g. riluzole).

In another embodiment, albumin fusion proteins and/or polynucleotides ofthe invention are administered in combination with vasodilating agentsand/or calcium channel blocking agents. Vasodilating agents that may beadministered with the albumin fusion proteins and/or polynucleotides ofthe invention include, but are not limited to, Angiotensin ConvertingEnzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril,captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril,moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril,and nylidrin), and nitrates (e., isosorbide dinitrate, isosorbidemononitrate, and nitroglycerin). Examples of calcium channel blockingagents that may be administered in combination with the albumin fusionproteins and/or polynucleotides of the invention include, but are notlimited to amlodipine, bepridil, diltiazem, felodipine, flunarizine,isradipine, nicardipine, nifedipine, nimodipine, and verapamil.

In certain embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withtreatments for gastrointestinal disorders. Treatments forgastrointestinal disorders that may be administered with the albuminfusion protein and/or polynucleotide of the invention include, but arenot limited to, H, histamine receptor antagonists (e.g., TAGAMET™(cimetidine), ZANTAC™ (ranitidine), PEPCID™ (famotidine), and AXID™(nizatidine)); inhibitors of H⁺, K⁺ATPase (e.g., PREVACID™(lansoprazole) and PRILOSEC™ (omeprazole)); Bismuth compounds (e.g.,PEPTO-BISMOL™ (bismuth subsalicylate) and DE-NOL™ (bismuth subcitrate));various antacids; sucralfate; prostaglandin analogs (e.g. CYTOTEC™(misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g.,surfactant laxatives, stimulant laxatives, saline and osmoticlaxatives); antidiarrheal agents (e.g., LOMOTIL™ (diphenoxylate),MOTOFEN™ (diphenoxin), and IMODIUM™ (loperamide hydrochloride)),synthetic analogs of somatostatin such as SANDOSTATIN™ (octreotide),antiemetic agents (e.g., ZOFRAN™ (ondansetron), KYTRIL™ (granisetronhydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine,perphenazine, prochlorperazine, promethazine, thiethylperazine,triflupromazine, domperidone, haloperidol, droperidol,trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, andnabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide andchlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholicacid; and pancreatic enzyme preparations such as pancreatin andpancrelipase.

In additional embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withother therapeutic or prophylactic regimens, such as, for example,radiation therapy.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions comprising albumin fusion proteins of theinvention. Optionally associated with such container(s) can be a noticein the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

Gene Therapy

Constructs encoding albumin fusion proteins of the invention can be usedas a part of a gene therapy protocol to deliver therapeuticallyeffective doses of the albumin fusion protein. A preferred approach forin vivo introduction of nucleic acid into a cell is by use of a viralvector containing nucleic acid, encoding an albumin fusion protein ofthe invention. Infection of cells with a viral vector has the advantagethat a large proportion of the targeted cells can receive the nucleicacid. Additionally, molecules encoded within the viral vector, e.g., bya cDNA contained in the viral vector, are expressed efficiently in cellswhich have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous nucleicacid molecules encoding albumin fusion proteins in vivo. These vectorsprovide efficient delivery of nucleic acids into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. The development of specialized cell lines (termed“packaging cells”) which produce only replication-defective retroviruseshas increased the utility of retroviruses for gene therapy, anddefective retroviruses are characterized for use in gene transfer forgene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:271). A replication defective retrovirus can be packaged into virionswhich can be used to infect a target cell through the use of a helpervirus by standard techniques. Protocols for producing recombinantretroviruses and for infecting cells in vitro or in vivo with suchviruses can be found in Current Protocols in Molecular Biology, Ausubel,F. M. et al.,. (eds.) Greene Publishing Associates, (1989), Sections9.10-9.14 and other standard laboratory manuals.

Another viral gene delivery system useful in the present invention usesadenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See, for example, Berkner et al.,BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434(1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 d1324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known tothose skilled in the art. Recombinant adenoviruses can be advantageousin certain circumstances in that they are not capable of infectingnondividing cells and can be used to infect a wide variety of celltypes, including epithelial cells (Rosenfeld et al., (1992) citedsupra). Furthermore, the virus particle is relatively stable andamenable to purification and concentration, and as above, can bemodified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al., cited supra;Haj-Ahmand et al., J. Virol. 57:267 (1986)).

In another embodiment, non-viral gene delivery systems of the presentinvention rely on endocytic pathways for the uptake of the subjectnucleotide molecule by the targeted cell. Exemplary gene deliverysystems of this type include liposomal derived systems, poly-lysineconjugates, and artificial viral envelopes. In a representativeembodiment, a nucleic acid molecule encoding an albumin fusion proteinof the invention can be entrapped in liposomes bearing positive chargeson their surface (e.g., lipofectins) and (optionally) which are taggedwith antibodies against cell surface antigens of the target tissue(Mizuno et al. (1992). No Shinkei Geka 20:547-5 5 1; PCT publicationWO91/06309; Japanese patent application 1WO7381; and European patentpublication EP-A43075).

Gene delivery systems for a gene encoding an albumin fusion protein ofthe invention can be introduced into a patient by any of a number ofmethods. For instance, a pharmaceutical preparation of the gene deliverysystem can be introduced systemically, e.g. by intravenous injection,and specific transduction of the protein in the target cells occurspredominantly from specificity of transfection provided by the genedelivery vehicle, cell-type or tissue-type expression due to thetranscriptional regulatory sequences controlling expression of thereceptor gene, or a combination thereof. In other embodiments, initialdelivery of the recombinant gene is more limited with introduction intothe animal being quite localized. For example, the gene delivery vehiclecan be introduced by catheter (see U.S. Pat. No. 5,328,470) or byStereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3 054-3 05 7).The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Where the albumin fusion protein can be produced intact fromrecombinant cells, e.g. retroviral vectors, the pharmaceuticalpreparation can comprise one or more cells which produce the albuminfusion protein.

Additional Gene Therapy Methods

Also encompassed by the invention are gene therapy methods for treatingor preventing disorders, diseases and conditions. The gene therapymethods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofan albumin fusion protein of the invention. This method requires apolynucleotide which codes for an albumin fusion protein of the presentinvention operatively linked to a promoter and any other geneticelements necessary for the expression of the fusion protein by thetarget tissue. Such gene therapy and delivery techniques are known inthe art, see, for example, WO90/11092, which is herein incorporated byreference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide encoding an albumin fusion protein of the presentinvention ex vivo, with the engineered cells then being provided to apatient to be treated with the fusion protein of the present invention.Such methods are well-known in the art. For example, see Belldegrun, A.,et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al.,Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J.Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60:221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990);Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato,L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al.,Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated byreference. In one embodiment, the cells which are engineered arearterial cells. The arterial cells may be reintroduced into the patientthrough direct injection to the artery, the tissues surrounding theartery, or through catheter injection.

As discussed in more detail below, the polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The polynucleotideconstructs may be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

In one embodiment, polynucleotides encoding the albumin fusion proteinsof the present invention is delivered as a naked polynucleotide. Theterm “naked” polynucleotide, DNA or RNA refers to sequences that arefree from any delivery vehicle that acts to assist, promote orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, polynucleotides encoding the albumin fusion proteinsof the present invention can also be delivered in liposome formulationsand lipofectin formulations and the like can be prepared by methods wellknown to those skilled in the art. Such methods are described, forexample, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, whichare herein incorporated by reference.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Appropriatevectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; andpEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Othersuitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of the polynucleotide sequence. Suitablepromoters include adenoviral promoters, such as the adenoviral majorlate promoter; or heterologous promoters, such as the cytomegalovirus(CMV) promoter; the respiratory syncytial virus (RSV) promoter;inducible promoters, such as the MMT promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTRs; theb-actin promoter; and human growth hormone promoters. The promoter alsomay be the native promoter for the gene corresponding to the Therapeuticprotein portion of the albumin fusion proteins of the invention.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within the an animal, including of muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney; gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising, thesecells. They are preferably delivered to and expressed in persistent,non-dividing, cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the polynucleotide constructs are complexed in aliposome preparation. Liposomal preparations for use in the instantinvention include cationic (positively charged), anionic (negativelycharged) and neutral preparations. However, cationic liposomes areparticularly preferred because a tight charge complex can be formedbetween the cationic liposome and the polyanionic nucleic acid. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416,which is herein incorporated by reference); mRiNA (Malone et al., Proc.Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporatedby reference); and purified transcription factors (Debs et al., J. Biol.Chem. (1990) 265:10189-10192, which is herein incorporated byreference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethyl ammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using, techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed SUVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys.Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978);Schaefer-Ridder et al., Science 215:166 (1982)), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 provide methodsfor delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingan albumin fusion protein of the present invention. Retroviruses fromwhich the retroviral plasmid vectors may be derived include, but are notlimited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Roussarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon apeleukemia virus, human immunodeficiency virus, Myeloproliferative SarcomaVirus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding an albumin fusion protein of thepresent invention. Such retroviral vector particles then may beemployed, to transduce eukaryotic cells, either in vitro or in vivo. Thetransduced eukaryotic cells will express a fusion protin of the presentinvention.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with polynucleotide contained in an adenovirus vector. Adenovirus can bemanipulated such that it encodes and expresses fusion protein of thepresent invention, and at the same time is inactivated in terms of itsability to replicate in a normal lytic viral life cycle: Adenovirusexpression is achieved without integration of the viral DNA into thehost cell chromosome, thereby alleviating concerns about insertionalmutagenesis. Furthermore, adenoviruses have been used as live entericvaccines for many years with an excellent safety profile (Schwartz etal. Am. Rev. Respir. Dis. 109:233-238 (1974)). Finally, adenovirusmediated gene transfer has been demonstrated in a number of instancesincluding transfer of alpha-1-antitrypsin and CFTR to the lungs ofcotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434;Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2; other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few-viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about. 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructis inserted into the AAV vector using standard cloning methods, such asthose found in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (1989). The recombinant AAV vector is thentransfected into packaging cells which are infected with a helper virus,using any standard technique, including lipofection, electroporation,calcium phosphate precipitation, etc. Appropriate helper viruses includeadenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.Once the packaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct. These viral particles are then used to transduce eukaryoticcells, either ex vivo or in vivo. The transduced cells will contain thepolynucleotide construct integrated into its genome, and will express afsuion protein of the invention.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding a polypeptide of the present invention) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication No. WO 96/29411, published Sep. 26, 1996;International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), which are hereinencorporated by reference. This method involves the activation of a genewhich is present in the target cells, but which is not normallyexpressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the desired endogenous polynucleotide sequence so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 3′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3‘end’ of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

The polynucleotide encoding an albumin fusion protein of the presentinvention may contain a secretory signal sequence that facilitatessecretion of the protein. Typically, the signal sequence is positionedin the coding region of the polynucleotide to be expressed towards or atthe 5′ end of the coding region. The signal sequence may be homologousor heterologous to the polynucleotide of interest and may be homologousor heterologous to the cells to be transfected. Additionally, the signalsequence may be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, an albumin fusion protein of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includefusion proteins of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site. In specificembodiments, suitable delivery vehicles for use with systemicadministration comprise liposomes comprising albumin fusion proteins ofthe invention for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling, et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Albumin fusion proteins of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Biological Activities

Albumin fusion proteins and/or polynucleotides encoding albumin fusionproteins of the present invention, can be used in assays to test for oneor more biological activities. If an albumin fusion protein and/orpolynucleotide exhibits an activity in a particular assay, it is likelythat the Therapeutic protein corresponding to the fusion portein may beinvolved in the diseases associated with the biological activity. Thus,the fusion protein could be used to treat the associated disease.

Members of the secreted family of proteins are believed to be involvedin biological activities associated with, for example, cellularsignaling. Accordingly, albumin fusion proteins of the invention andpolynucleotides encoding these protiens, may be used in diagnosis,prognosis, prevention and/or treatment of diseases and/or disordersassociated with aberrant activity of secreted polypeptides.

In preferred embodiments, fusion proteins of the present invention maybe used in the diagnosis, prognosis, prevention and/or treatment ofdiseases and/or disorders relating to diseases and disorders of theendocrine system, the nervous system (See, for example, “NeurologicalDisorders” section below), the immune system (See, for example, “ImmuneActivity” section below), respiratory system (See, for example,“Respiratory Disorders” section below), cardiovascular system (See, forexample, “Cardiovascular Disorders” section below), reproductive system(See, for example, “Reproductive System Disorders” section below)digestive system (See, for example, “Gastrointestinal Disorders” sectionbelow), diseases and/or disorders relating to cell proliferation (See,for example, “Hyperproliferative Disorders/Cancer” section below),and/or diseases or disorders relating to the blood ((See, for example,“Blood-Related Disorders” section below).

In preferred embodiments, the present invention encompasses a method oftreating a disease or disorder listed in the “Preferred Indication Y”column of Table 1 comprising administering to a patient in which suchtreatment, prevention or amelioration is desired an albumin fusionprotein of the invention that comprises a Therapeutic protein portioncorresponding to a Therapeutic protein disclosed in the “TherapeuticProtein X” column of Table 1 (in the same row as the disease or disorderto be treated is listed in the “Preferred Indication Y” column ofTable 1) in an amount effective to treat prevent or ameliorate thedisease or disorder.

In certain embodiments, an albumin fusion protein of the presentinvention may be used to diagnose and/or pro-nose diseases and/ordisorders associated with the tissue(s) in which the gene correspondingto the Therapeutic protein portion of the fusion portien of theinvention is expressed.

Thus, fusion proteins of the invention and polynucleotides encodingalbumin fusion proteins of the invention are useful in the diagnosis,detection and/or treatment of diseases and/or disorders associated withactivities that include, but are not limited to, prohormone activation,neurotransmitter activity, cellular signaling, cellular proliferation,cellular differentiation, and cell migration.

More generally, fusion proteins of the invention and polynucleotidesencoding albumin fusion proteins of the invention may be useful for thediagnosis, prognosis, prevention and/or treatment of diseases and/ordisorders associated with the following systems.

Immune Activity

Albumin fusion proteins of the invention and polynucleotides encodingalbumin fusion proteins of the invention may be useful in treating,preventing, diagnosing and/or prognosing diseases, disorders, and/orconditions of the immune system, by, for example, activating orinhibiting the proliferation, differentiation, or mobilization(chemotaxis) of immune cells. Immune cells develop through a processcalled hematopoiesis, producing myeloid (platelets, red blood cells,neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cellsfrom pluripotent stem cells. The etiology of these immune diseases,disorders, and/or conditions may be genetic, somatic, such as cancer andsome autoimmune diseases, acquired (e.g., by chemotherapy or toxins), orinfectious. Moreover, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention can beused as a marker or detector of a particular immune system disease ordisorder.

In another embodiment, a fusion protein of the invention and/orpolynucleotide encoding an albumin fusion protein of the invention, maybe used to treat diseases and disorders of the immune system and/or toinhibit or enhance an immune response generated by cells associated withthe tissue(s) in which the polypeptide of the invention is expressed.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be useful in treating,preventing, diagnosing, and/or prognosing immunodeficiencies, includingboth congenital and acquired immunodeficiencies. Examples of B cellimmunodeficiencies in which immunoglobulin levels B cell function and/orB cell numbers are decreased include: X-linked agammaglobulinemia(Bruton's disease), X-linked infantile agammaglobulinemia, X-linkedimmunodeficiency with hyper IgM, non X-linked immunodeficiency withhyper IgM, X-linked lymphoproliferative syndrome (XLP),agammaglobulinemia including congenital and acquired agammaglobulinemia;adult onset agammaglobulinemia, late-onset agammaglobulinemia,dysgammaglobulinemia, hypogammaglobulinemia, unspecifiedhypogammaglobulinemia, recessive agammaglobulinemia (Swiss type),Selective IgA deficiency, selective IgA deficiency, selective IgGsubclass deficiencies, IgG subclass deficiency (with or without IgAdeficiency), Ig deficiency with increased IgM, IgG and IgA deficiencywith increased IgM, antibody deficiency with normal or elevated Igs, Igheavy chain deletions, kappa chain deficiency, B celllymphoproliferative disorder (BLPD), common variable immunodeficiency(CVID), common variable immunodeficiency (CVI) (acquired), and transienthypogammaglobulinemia of infancy.

In specific embodiments, ataxia-telangiectasia or conditions associatedwith ataxia-telangiectasia are treated, prevented, diagnosed, and/orprognosing using the fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention.

Examples of congenital immunodeficiencies in which T cell and/or B cellfunction and/or number is decreased include, but are not limited to:DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including,but not limited to, X-linked SCID, autosomal recessive SCID, adenosinedeaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency,Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrichsyndrome, and ataxia telangiectasia), thymic hypoplasia, third andfourth pharyngeal pouch syndrome, 22q11.2 deletion, chronicmucocutaneous candidiasis, natural killer cell deficiency (NK),idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant Tcell defect (unspecified), and unspecified immunodeficiency of cellmediated immunity.

In specific embodiments, DiGeorge anomaly or conditions associated withDiGeorge anomaly are treated, prevented, diagnosed, and/or prognosedusing fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention.

Other immunodeficiencies that may be treated, prevented, diagnosed,and/or pro-nosed using fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention,include, but are not limited to, chronic granulomatous disease,Chédiak-Higashi syndrome, myeloperoxidase deficiency, leukocyteglucose-6-phosphate dehydrogenase deficiency, X-linkedlymphoproliferative syndrome (XLP), leukocyte adhesion deficiency,complement component deficiencies (including C1, C2, C3, C4, C5, C6, C7,CS and/or C9 deficiencies), reticular dysgenesis, thymicalymphoplasia-aplasia, immunodeficiency with thymoma, severe congenitalleukopenia, dysplasia with immunodeficiency, neonatal neutropenia, shortlimbed dwarfism, and Nezelof syndrome-combined immunodeficiency withIgs.

In a preferred embodiment, the immunodeficiencies and/or conditionsassociated with the immunodeficiencies recited above are treated,prevented, diagnosed and/or prognosed using fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention.

In a preferred embodiment fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention couldbe used as an agent to boost immunoresponsiveness among immunodeficientindividuals. In specific embodiments, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventioncould be used as an agent to boost immunoresponsiveness among B celland/or T cell immunodeficient individuals.

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful intreating, preventing, diagnosing and/or prognosing autoimmune disorders.Many autoimmune disorders result from inappropriate recognition of selfas foreign material by immune cells. This inappropriate recognitionresults in an immune response leading to the destruction of the hosttissue. Therefore, the administration of fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention that can inhibit an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

Autoimmmune diseases or disorders that may be treated, prevented,diagnosed and/or pro-nosed by fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to, one or more of the following: systemiclupus erythematosus, rheumatoid arthritis, ankylosing spondylitis,multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis,autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia,autoimmune thrombocytopenia purpura, autoimmune neonatalthrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g.,Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome,Pemphigus vulgaris, myasthenia gravis, Grave's disease(hyperthyroidism), and insulin-resistant diabetes mellitus.

Additional disorders that are likely to have an autoimmune componentthat may be treated, prevented, and/or diagnosed with the albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention include, but are not limited to, type IIcollagen-induced arthritis, antiphospholipid syndrome, dermatitis,allergic encephalomyelitis, myocarditis, relapsing polychondritis,rheumatic heart disease, neuritis, uveitis ophthalmia,polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmunepulmonary inflammation, autism, Guillain-Barre Syndrome, insulindependent diabetes mellitus, and autoimmune inflammatory eye disorders.

Additional disorders that are likely to have an autoimmune componentthat may be treated, prevented, diagnosed and/or prognosed with thealbumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include, but are not limitedto, scleroderma with anti-collagen antibodies (often characterized,e.g., by nucleolar and other nuclear antibodies), mixed connectivetissue disease (often characterized, e.g., by antibodies to extractablenuclear antigens (e.g., ribonucleoprotein)), polymyositis (oftencharacterized, e.g., by nonhistone ANA), pernicious anemia (oftencharacterized, e.g., by antiparietal cell, microsomes, and intrinsicfactor antibodies), idiopathic Addison's disease (often characterized,e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility(often characterized, e.g., by antispermatozoal antibodies),glomerulonephritis (often characterized, e.g., by glomerular basementmembrane antibodies or immune complexes), bullous pemphigoid (oftencharacterized, e.g., by IgG and complement in basement membrane),Sjogren's syndrome (often characterized e.g., by multiple tissueantibodies, and/or a specific nonhistone ANA (SS-B)), diabetes-mellitus(often characterized, e.g., by cell-mediated and humoral islet cellantibodies), and adrenergic drug resistance (including adrenergic drugresistance with asthma or cystic fibrosis) (often characterized, e.g.,by beta-adrenergic receptor antibodies).

Additional disorders that may have an autoimmune component that may betreated, prevented, diagnosed and/or prognosed with the albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention include, but are not limited to, chronicactive hepatitis (often characterized, e.g., by smooth muscleantibodies), primary biliary cirrhosis (often characterized, e.g., bymitochondria antibodies), other endocrine gland failure (oftencharacterized, e.g., by specific tissue antibodies in some cases),vitiligo (often characterized, e.g., by melanocyte antibodies),vasculitis (often characterized, e.g., by Ig and complement in vesselwalls and/or low serum complement), post-MI (often characterized, e.g.,by myocardial antibodies), cardiotomy syndrome (often characterized,e.g., by myocardial antibodies), urticaria (often characterized, e.g.,by IgG and IgM antibodies to IgE), atopic dermatitis (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), asthma (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), and many otherinflammatory, granulomatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, diagnosed and/or pro-nosed using for example, fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention. In a specific preferred embodiment,rheumatoid arthritis is treated, prevented, and/or diagnosed usingfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention.

In another specific preferred embodiment, systemic lupus erythematosusis treated, prevented, and/or diagnosed using fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention. In another specific preferred embodiment, idiopathicthrombocytopenia purpura is treated, prevented, and/or diagnosed usingfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention.

In another specific preferred embodiment IgA nephropathy is treated,prevented, and/or diagnosed using fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, diagnosed and/or pro-nosed using fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention.

In preferred embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused as a immunosuppressive agent(s).

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be useful in treating,preventing, prognosing, and/or diagnosing diseases, disorders, and/orconditions of hematopoietic cells. Albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention could be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with a decrease in certain (or many) types hematopoieticcells, including but not limited to, leukopenia, neutropenia, anemia,and thrombocytopenia. Alternatively, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventioncould be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with an increase in certain (or many) types of hematopoieticcells, including but not limited to, histiocytosis.

Allergic reactions and conditions, such as asthma (particularly allergicasthma) or other respiratory problems, may also be treated, prevented,diagnosed and/or prognosed using fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention.Moreover, these molecules can be used to treat, prevent, prognose,and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule,or blood group incompatibility.

Additionally, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may be used to treat,prevent, diagnose and/or prognose IgE-mediated allergic reactions. Suchallergic reactions include, but are not limited to, asthma, rhinitis,and eczema. In specific embodiments, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used to modulate IgE concentrations in vitro or in vivo.

Moreover, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention have uses in thediagnosis, prognosis, prevention, and/or treatment of inflammatoryconditions. For example, since fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayinhibit the activation, proliferation and/or differentiation of cellsinvolved in an inflammatory response, these molecules can be used toprevent and/or treat chronic and acute inflammatory conditions. Suchinflammatory conditions include, but are not limited to, for example,inflammation associated with infection (e.g., septic shock, sepsis, orsystemic inflammatory response syndrome), ischemia-reperfusion injury,endotoxin lethality, complement-mediated hyperacute rejection,nephritis, cytokine or chemokine induced lung injury, inflammatory boweldisease, Crohn's disease, over production of cytokines (e.g., TNF orIL-1.), respiratory disorders (e.g., asthma and allergy);gastrointestinal disorders (e.g., inflammatory bowel disease); cancers(e.g., gastric, ovarian, lung, bladder, liver, and breast); CNSdisorders (e.g., multiple sclerosis; ischemic brain injury and/orstroke, traumatic brain injury, neurodegenerative disorders (e.g.,Parkinson's disease and Alzheimer's disease); AIDS-related dementia; andprion disease); cardiovascular disorders (e.g., atherosclerosis,myocarditis, cardiovascular disease, and cardiopulmonary bypasscomplications); as well as many additional diseases, conditions, anddisorders that are characterized by inflammation (e.g., hepatitis,rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis,dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemiclupus erythematosus, diabetes mellitus, and allogenic transplantrejection).

Because inflammation is a fundamental defense mechanism, inflammatorydisorders can effect virtually any tissue of the body. Accordingly,fusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention, have uses in the treatment oftissue-specific inflammatory disorders, including, but not limited to,adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis,blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis,cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis,dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis,eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis,gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis,laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis,mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis,orchitis, osteochondritis, otitis, pericarditis, peritendonitis,peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis,pulpitis, retinitis, rhinitis, salpingitis, scleritis,sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis,stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis,and vaginitis.

In specific embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, areuseful to diagnose, prognose, prevent, and/or treat organ transplantrejections and graft-versus-host disease. Organ rejection occurs by hostimmune cell destruction of the transplanted tissue through an immuneresponse. Similarly, an immune response is also involved in GVHD, but,in this case, the foreign transplanted immune cells destroy the hosttissues. Polypeptides, antibodies, or polynucleotides of the invention,and/or agonists or antagonists thereof, that inhibit an immune response,particularly the activation, proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD. In specific embodiments, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention, that inhibit an immune response, particularly the activationproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing experimental allergic and hyperacutexenograft rejection.

In other embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, areuseful to diagnose, prognose, prevent, and/or treat immune complexdiseases, including, but not limited to, serum sickness, poststreptococcal glomerulonephritis, polyarteritis nodosa, and immunecomplex-induced vasculitis.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention can be used to treat, detect,and/or prevent infectious agents. For example, by increasing the immuneresponse, particularly increasing the proliferation activation and/ordifferentiation of B and/or T cells, infectious diseases may be treated,detected, and/or prevented. The immune response may be increased byeither enhancing an existing immune response, or by initiating a newimmune response. Alternatively, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayalso directly inhibit the infectious agent (refer to section ofapplication listing infectious agents, etc), without necessarilyeliciting an immune response.

In another embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused as a vaccine adjuvant that enhances immune responsiveness to anantigen. In a specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an adjuvant to enhance tumor-specific immuneresponses.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an adjuvant to enhance anti-viral immune responses.Anti-viral immune responses that may be enhanced using the compositionsof the invention as an adjuvant, include virus and virus associateddiseases or symptoms described herein or otherwise known in the art. Inspecific embodiments, the compositions of the invention are used as anadjuvant to enhance an immune response to a virus, disease, or symptomselected from the group consisting of: AIDS, meningitis, Dengue, EBV,and hepatitis (e.g., hepatitis B). In another specific embodiment, thecompositions of the invention are used as an adjuvant to enhance animmune response to a virus, disease, or symptom selected from the groupconsisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus,Japanese B encephalitis, influenza A and B, parainfluenza, measles,cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpessimplex, and yellow fever.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an adjuvant to enhance anti-bacterial or anti-fungal immuneresponses. Anti-bacterial or anti-fungal immune responses that may beenhanced using the compositions of the invention as an adjuvant, includebacteria or fungus and bacteria or fungus associated diseases orsymptoms described herein or otherwise known in the art. In specificembodiments, the compositions of the invention are used as an adjuvantto enhance an immune response to a bacteria or fungus, disease, orsymptom selected from the group consisting of: tetanus, Diphtheria,botulism, and meningitis type B.

In another specific embodiment, the compositions of the invention areused as an adjuvant to enhance an immune response to a bacteria orfungus, disease, or symptom selected from the group consisting of:Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonellaparatyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group Bstreptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, and Borrelia burgdorferi.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an adjuvant to enhance anti-parasitic immune responses.Anti-parasitic immune responses that may be enhanced using thecompositions of the invention as an adjuvant, include parasite andparasite associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium (malaria) or Leishmania.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay also be employed to treat infectious diseases including silicosis,sarcoidosis, and idiopathic pulmonary fibrosis; for example, bypreventing the recruitment and activation of mononuclear phagocytes.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an antigen for the generation of antibodies to inhibit orenhance immune mediated responses against polypeptides of the invention.

In one embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areadministered to an animal (e.g., mouse, rat, rabbit, hamster, guineapig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,non-human primate, and human, most preferably human) to boost the immunesystem to produce increased quantities of one or more antibodies (e.g.,IgG, IgA, IgM, and IgE), to induce higher affinity antibody productionand immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE),and/or to increase an immune response.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a stimulator of B cell responsiveness to pathogens.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an activator of T cells.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an agent that elevates the immune status of an individualprior to their receipt of immunosuppressive therapies.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an agent to induce higher affinity antibodies.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an agent to increase serum immunoglobulin concentrations.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an agent to accelerate recovery of immunocompromisedindividuals.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an agent to boost immunoresponsiveness among agedpopulations and/or neonates.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an immune system enhancer prior to, during, or after bonemarrow transplant and/or other transplants (e.g., allogeneic orxenogeneic organ transplantation). With respect to transplantation,compositions of the invention may be administered prior to, concomitantwith, and/or after transplantation. In a specific embodiment,compositions of the invention are administered after transplantation,prior to the beginning of recovery of T-cell populations. In anotherspecific embodiment, compositions of the invention are firstadministered after transplantation after the beginning of recovery of Tcell populations, but prior to full recovery of B cell populations.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an agent to boost immunoresponsiveness among individualshaving an acquired loss of B cell function. Conditions resulting in anacquired loss of B cell function that may be ameliorated or treated byadministering the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention,include, but are not limited to, HIV Infection, AIDS, bone marrowtransplant, and B cell chronic lymphocytic leukemia (CLL).

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an agent to boost immunoresponsiveness among individualshaving a temporary immune deficiency. Conditions resulting in atemporary immune deficiency that may be ameliorated or treated byadministering the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention,include, but are not limited to, recovery from viral infections (e.g.,influenza), conditions associated with malnutrition, recovery frominfectious mononucleosis, or conditions associated with stress, recoveryfrom measles, recovery from blood transfusion, and recovery fromsurgery.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a regulator of antigen presentation by monocytes, dendriticcells, and/or B-cells. In one embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention enhance antigen presentation or antagonizes antigenpresentation in vitro or in vivo. Moreover, in related embodiments, thisenhancement or antagonism of antigen presentation may be useful as ananti-tumor treatment or to modulate the immune system.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as an agent to direct an individual's immune system towardsdevelopment of a humoral response (i.e. TH2) as opposed to a TH1cellular response.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a stimulator of B cell production in pathologies such asAIDS, chronic lymphocyte disorder and/or Common VariableImmunodificiency.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a therapy for generation and/or regeneration of lymphoidtissues following surgery, trauma or genetic defect. In another specificembodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused in the pretreatment of bone marrow samples prior to transplant.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a gene-based therapy for genetically inherited disordersresulting in immuno-incompetence/immunodeficiency such as observed amongSCID patients.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a means of activating monocytes/macrophages to defendagainst parasitic diseases that effect monocytes such as Leishmania.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a means of regulating secreted cytokines that are elicitedby polypeptides of the invention.

In another embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused in one or more of the applications described herein, as they mayapply to veterinary medicine.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a means of blocking various aspects of immune responses toforeign agents or self. Examples of diseases or conditions in whichblocking of certain aspects of immune responses may be desired includeautoimmune disorders such as lupus, and arthritis, as well asimmunoresponsiveness to skin allergies, inflammation, bowel disease,injury and diseases/disorders associated with pathogens.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a therapy for preventing the B cell proliferation and Igsecretion associated with autoimmune diseases such as idiopathicthrombocytopenic purpura, systemic lupus erythematosus and multiplesclerosis.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention invention are used as a inhibitor of B and/orT cell migration in endothelial cells. This activity disrupts tissuearchitecture or cognate responses and is useful, for example indisrupting immune responses, and blocking sepsis.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a therapy for chronic hypergammaglobulinemia evident in suchdiseases as monoclonal gammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonal gammopathies, andplasmacytomas.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be employed for instance to inhibit polypeptide chemotaxis andactivation of macrophages and their precursors, and of neutrophils,basophils, B lymphocytes and some T-cell subsets, e.g., activated andCD8 cytotoxic T cells and natural killer cells, in certain autoimmuneand chronic inflammatory and infective diseases. Examples of autoimmunediseases are described herein and include multiple sclerosis, andinsulin-dependent diabetes.

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may also be employedto treat idiopathic hyper-eosinophilic syndrome by, for example,preventing eosinophil production and migration.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used to enhance or inhibit complement mediated cell lysis.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used to enhance or inhibit-antibody dependent cellular cytotoxicity.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay also be employed for treating atherosclerosis, for example, bypreventing monocyte infiltration in the artery wall.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be employed to treat adult respiratory distress syndrome (ARDS).

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful for stimulating wound and tissue repair, stimulatingangiogenesis, and/or stimulating the repair of vascular or lymphaticdiseases or disorders. Additionally, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used to stimulate the regeneration of mucosal surfaces.

In a specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used to diagnose, prognose, treat, and/or prevent a disordercharacterized by primary or acquired immunodeficiency, deficient serumimmunoglobulin production, recurrent infections, and/or immune systemdysfunction. Moreover, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused to treat or prevent infections of the joints, bones, skin, and/orparotid glands, blood-borne infections (e.g., sepsis, meningitis, septicarthritis, and/or osteomyelitis), autoimmune diseases (e.g., thosedisclosed herein), inflammatory disorders, and malignancies, and/or anydisease or disorder or condition associated with these infections,diseases, disorders and/or malignancies) including, but not limited to,CVID, other primary immune deficiencies, HIV disease, CLL, recurrentbronchitis, sinusitis, otitis media, conjunctivitis, pneumonia,hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster),and/or pneumocystis camii. Other diseases and disorders that may beprevented, diagnosed, prognosed, and/or treated with fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention include, but are not limited to, HIV infection, HTLV-BLVinfection, lymphopenia, phagocyte bactericidal dysfunction anemia,thrombocytopenia, and hemoglobinuria.

In another embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to treat, and/or diagnose an individual having common variableimmunodeficiency disease (“CVID”; also known as “acquiredacammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset ofthis disease.

In a specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used to diagnose, prognose, prevent, and/or treat cancers orneoplasms including immune cell or immune tissue-related cancers orneoplasms. Examples of cancers or neoplasms that may be prevented,diagnosed, or treated by fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to, acute myelogenous leukemia, chronicmyelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acutelymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas,multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/ordiseases and disorders described in the section entitled“Hyperproliferative Disorders” elsewhere herein.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a therapy for decreasing cellular proliferation of LargeB-cell Lymphomas.

In another specific embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a means of decreasing the involvement of B cells and Ivassociated with Chronic Myelogenous Leukemia.

In specific embodiments, the compositions of the invention are used asan agent to boost immunoresponsiveness among B cell immunodeficientindividuals, such as, for example, an individual who has undergone apartial or complete splenectomy.

Blood-Related Disorders

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be used tomodulate hemostatic (the stopping of bleeding) or thrombolytic (clotdissolving) activity. For example, by increasing hemostatic orthrombolytic activity, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention couldbe used to treat or prevent blood coagulation diseases, disorders,and/or conditions (e.g., afibrinogenemia, factor deficiencies,hemophilia), blood platelet diseases, disorders, and/or conditions(e.g., thrombocytopenia), or wounds resulting from trauma, surgery, orother causes. Alternatively, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention thatcan decrease hemostatic or thrombolytic activity could be used toinhibit or dissolve clotting These molecules could be important in thetreatment or prevention of heart attacks (infarction), strokes, orscarring.

In specific embodiments, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used to prevent, diagnose, pro-nose, and/or treat thrombosis,arterial thrombosis, venous thrombosis, thromboembolism, pulmonaryembolism, atherosclerosis, myocardial infarction, transient ischemicattack, unstable angina. In specific embodiments, the albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention may be used for the prevention of occulsion ofsaphenous grafts, for reducing the risk of periprocedural thrombosis asmight accompany angioplasty procedures, for reducing the risk of strokein patients with atrial fibrillation including nonrheumatic atrialfibrillation, for reducing the risk of embolism associated withmechanical heart valves and or mitral valves disease. Other uses for thealbumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, include, but are not limitedto, the prevention of occlusions in extrcorporeal devices (e.g.,intravascular canulas, vascular access shunts in hemodialysis patients,hemodialysis machines, and cardiopulmonary bypass machines).

In another embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, maybe used to prevent, diagnose, prognose, and/or treat diseases anddisorders of the blood and/or blood forming organs associated with thetissue(s) in which the polypeptide of the invention is expressed.

The fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be used to modulatehematopoietic activity (the formation of blood cells). For example, thealbumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be used to increase thequantity of all or subsets of blood cells, such as, for example,erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g.,basophils, eosinophils, neutrophils, mast cells, macrophages) andplatelets. The ability to decrease the quantity of blood cells orsubsets of blood cells may be useful in the prevention, detection,diagnosis and/or treatment of anemias and leukopenias described below.Alternatively, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused to decrease the quantity of all or subsets of blood cells, such asfor example, erythrocytes, lymphocytes (B or T cells), myeloid cells(e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) andplatelets. The ability to decrease the quantity of blood cells orsubsets of blood cells may be useful in the prevention, detection,diagnosis and/or treatment of leukocytoses, such as, for exampleeosinophilia.

The fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be used to prevent, treat,or diagnose blood dyscrasia.

Anemias are conditions in which the number of red blood cells or amountof hemoglobin (the protein that carries oxygen) in them is below normal.Anemia may be caused by excessive bleeding, decreased red blood cellproduction, or increased red blood cell destruction (hemolysis). Thealbumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be useful in treating,preventing, and/or diagnosing anemias. Anemias that may be treatedprevented or diagnosed by the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventioninclude iron deficiency anemia, hypochromic anemia, microcytic anemia,chlorosis, hereditary siderob;astic anemia, idiopathic acquiredsideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g.,pernicious anemia, (vitamin B12 deficiency) and folic acid deficiencyanemia), aplastic anemia, hemolytic anemias (e.g., autoimmune helolyticanemia, microangiopathic hemolytic anemia, and paroxysmal nocturnalhemoglobinuria). The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in treating, preventing, and/or diagnosing anemias associatedwith diseases including but not limited to, anemias associated withsystemic lupus erythematosus, cancers, lymphomas, chronic renal disease,and enlarged spleens. The albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful in treating, preventing, and/or diagnosing anemias arisingfrom drug treatments such as anemias associated with methyldopa dapsone,and/or sulfadrugs. Additionally, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in treating, preventing, and/or diagnosing anemias associatedwith abnormal red blood cell architecture including but not limited to,hereditary spherocytosis, hereditary elliptocytosis, glucose-6phosphatedehydrogenase deficiency, and sickle cell anemia.

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful intreating, preventing, and/or diagnosing hemoglobin abnormalities, (e.g.,those associated with sickle cell anemia, hemoglobin C disease,hemoglobin S—C disease, and hemoglobin E disease). Additionally, thealbumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be useful in diagnosing,prognosing, preventing, and/or treating thalassemias, including, but notlimited to, major and minor forms of alpha-thalassemia andbeta-thalassemia.

In another embodiment, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful in diagnosing, prognosing, preventing, and/or treatingbleeding disorders including, but not limited to, thrombocytopenia(e.g., idiopathic thrombocytopenic purpura, and thromboticthrombocytopenic purpura), Von Willebrand's disease, hereditary plateletdisorders (e.g., storage pool disease such as Chediak-Higashi andHermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia,and Bernard-Soulier syndrome), hemolytic-uremic syndrome, hemopheliassuch as hemophelia A or Factor VII deficiency and Christmas disease orFactor IX deficiency, Hereditary Hemorhhagic Telangiectsia, also knownas Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonleinpurpura) and disseminated intravascular coagulation.

The effect of the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention on theclotting time of blood may be monitored using any of the clotting testsknown in the art including, but not limited to, whole blood partialthromboplastin time (PTT), the activated partial thromboplastin time(aPTT), the activated clotting time (ACT), the recalcified activatedclotting time, or the Lee-White Clotting time.

Several diseases and a variety of drugs can cause platelet dysfunction.Thus, in a specific embodiment, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in diagnosing, prognosing, preventing, and/ortreating acquired platelet dysfunction such as platelet dysfunctionaccompanying kidney failure, leukemia, multiple myeloma, cirrhosis ofthe liver, and systemic lupus erythematosus as well as plateletdysfunction associated with drug treatments, including treatment withaspirin ticlopidine, nonsteroidal anti-inflammatory drugs (used forarthritis, pain, and sprains), and penicillin in high doses.

In another embodiment, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful in diagnosing, prognosing, preventing, and/or treatingdiseases and disorders characterized by or associated with increased ordecreased numbers of white blood cells. Leukopenia occurs when thenumber of white blood cells decreases below normal. Leukopenias include,but are not limited to, neutropenia and lymphocytopenia. An increase inthe number of white blood cells compared to normal is known asleukocytosis. The body generates increased numbers of white blood cellsduring infection. Thus, leukocytosis may simply be a normalphysiological parameter that reflects infection. Alternatively,leukocytosis may be an indicator of injury or other disease such ascancer. Leokocytoses, include but are not limited to, eosinophilia, andaccumulations of macrophages. In specific embodiments, the albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention may be useful in diagnosing,prognosing, preventing, and/or treating leukopenia. In other specificembodiments, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in diagnosing, prognosing, preventing, and/or treatingleukocytosis.

Leukopenia may be a generalized decreased in all types of white bloodcells, or may be a specific depletion of particular types of white bloodcells. Thus, in specific embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in diagnosing, prognosing, preventing, and/ortreating decreases in neutrophil numbers, known as neutropenia.Neutropenias that may be diagnosed, prognosed, prevented, and/or treatedby the albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention include, but are notlimited to, infantile genetic agranulocytosis, familial neutropenia,cyclic neutropenia, neutropenias resulting, from or associated withdietary deficiencies (e.g., vitamin B 12 deficiency or folic aciddeficiency), neutropenias resulting from or associated with drugtreatments (e.g., antibiotic regimens such as penicillin treatment,sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs,anti-thyroid drugs, and cancer chemotherapy), and neutropenias resultingfrom increased neutrophil destruction that may occur in association withsome bacterial or viral infections, allergic disorders, autoimmunediseases, conditions in which an individual has an enlarged spleen(e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatmentregimens.

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful indiagnosing, prognosing, preventing, and/or treating lymphocytopenias(decreased numbers of B and/or T lymphocytes), including, but notlimited to, lymphocytopenias resulting from or associated with stress,drug treatments (e.g., drug treatment with corticosteroids, cancerchemotherapies, and/or radiation therapies), AIDS infection and/or otherdiseases such as, for example, cancer, rheumatoid arthritis, systemiclupus erythematosus, chronic infections, some viral infections and/orhereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome,severe combined immunodeficiency, ataxia telangiectsia).

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful indiagnosing, prognosing, preventing, and/or treating diseases anddisorders associated with macrophage numbers and/or macrophage functionincluding, but not limited to, Gaucher's disease, Niemann-Pick disease,Letterer-Siwe disease and Hand-Schuller-Christian disease.

In another embodiment, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful in diagnosing, prognosing, preventing, and/or treatingdiseases and disorders associated with eosinophil numbers and/oreosinophil function including, but not limited to, idiopathichypereosinophilic syndrome, eosinophilia-myalgia syndrome, andHand-Schuller-Christian disease.

In yet another embodiment, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful in diagnosing, prognosing, preventing, and/or treatingleukemias and lymphomas including, but not limited to, acute lymphocytic(lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous,myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia(e.g., B cell leukemias, T cell leukemias, Sezary syndrome, and Hairycell leukemia), chronic myelocytic (myeloid, myelogenous, orgranulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma,Burkitt's lymphoma, and mycosis fungoides.

In other embodiments, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful in diagnosing, prognosing, preventing, and/or treatingdiseases and disorders of plasma cells including, but not limited to,plasma cell dyscrasias, monoclonal gammaopathies, monoclonalgammopathies of undetermined significance, multiple myeloma,macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia,and Raynaud's phenomenon.

In other embodiments, the albumin fusion proteins of the inventionand/or polynucleotides encodina albumin fusion proteins of the inventionmay be useful in treating preventing, and/or diagnosingmyeloproliferative disorders, including but not limited to, polycythemiavera, relative polycythemia, secondary polycythemia, myelofibrosis,acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia,(including both primary and seconday thrombocythemia) and chronicmyelocytic leukemia.

In other embodiments, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful as a treatment prior to surgery, to increase blood cellproduction.

In other embodiments, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful as an agent to enhance the migration, phagocytosis,superoxide production, antibody dependent cellular cytotoxicity ofneutrophils, eosionophils and macrophages.

In other embodiments, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful as an agent to increase the number of stem cells incirculation prior to stem cells pheresis. In another specificembodiment, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful as an agent to increase the number of stem cells in circulationprior to platelet pheresis.

In other embodiments, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful as an agent to increase cytokine production.

In other embodiments, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful in preventing, diagnosing, and/or treating primaryhematopoietic disorders.

Hyperproliferative Disorders

In certain embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention can beused to treat or detect hyperproliferative disorders, includingneoplasms. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayinhibit the proliferation of the disorder through direct or indirectinteractions. Alternatively, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayproliferate other cells which can inhibit the hyperproliferativedisorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detectedby fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include, but are not limited toneoplasms located in the: colon, abdomen, bone, breast, digestivesystem, liver, pancreas, peritoneum, endocrine glands (adrenal,parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, headand neck, nervous (central and peripheral), lymphatic system, pelvis,skin, soft tissue, spleen, thorax, and urogenital tract.

Similarly, other hyperproliferative disorders can also be treated ordetected by fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention. Examples of suchhyperproliferative disorders include, but are not limited to: AcuteChildhood Lymphoblastic Leukemia, Acute. Lymphoblastic Leukemia, AcuteLymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma,Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer,Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, AdultHodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin'sLymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, IntestinalCancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet CellPancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer,Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, MetastaticPrimary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, MultipleMyeloma, Multiple Myeloma/Plasma Cell Neoplasm, MyelodysplasticSyndrome, Myelogenous Leukemia, Myeloid Leukemia, MyeloproliferativeDisorders, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult PrimaryMetastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/MalignantFibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,Osteosarcoma Malignant Fibrous Histiocytoma of Bone, Ovarian EpithelialCancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor,Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, PenileCancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer,Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis andUreter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell LungCancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine. Sarcoma, Vaginal Cancer, Visual Pathway and HypothalamicGlioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor,and any other hyperproliferative disease, besides neoplasia, located inan organ system listed above.

In another preferred embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to diagnose, prognose, prevent, and/or treatpremalignant conditions and to prevent progression to a neoplastic ormalignant state, including but not limited to those disorders describedabove. Such uses are indicated in conditions known or suspected ofpreceding progression to neoplasia or cancer, in particular, wherenon-neoplastic cell growth consisting of hyperplasia, metaplasia, ormost particularly, dysplasia has occurred (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79.)

Hyperplasia is a form of controlled cell proliferation, involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. Hyperplastic disorders which can bediagnosed, prognosed, prevented, and/or treated with fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention include; but are not limited to, angiofollicularmediastinal lymph node hyperplasia, angiolymphoid hyperplasia witheosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia,benign giant lymph node hyperplasia, cementum hyperplasia, congenitaladrenal hyperplasia, congenital sebaceous hyperplasia, cystichyperplasia, cystic hyperplasia of the breast, denture hyperplasia,ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia,focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibroushyperplasia, inflammatory papillary hyperplasia, intravascular papillaryendothelial hyperplasia, nodular hyperplasia of prostate, nodularregenerative hyperplasia, pseudoepitheliomatous hyperplasia, senilesebaceous hyperplasia, and verrucous hyperplasia.

Metaplasia is a form of controlled cell growth in which one type ofadult or fully differentiated cell substitutes for another type of adultcell. Metaplastic disorders which can be diagnosed, prognosed,prevented, and/or treated with fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to, agnogenic myeloid metaplasia, apocrinemetaplasia, atypical metaplasia, autoparenchymatous metaplasia,connective tissue metaplasia, epithelial metaplasia, intestinalmetaplasia, metaplastic anemia, metaplastic ossification, metaplasticpolyps, myeloid metaplasia, primary myeloid metaplasia, secondarymyeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion,and symptomatic myeloid metaplasia.

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation. Dysplastic disorders which can be diagnosed,prognosed, prevented, and/or treated with fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention include, but are not limited to, anhidrotic ectodermaldysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia,atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia,cervical dysplasia, chondroectodermal dysplasia, cleidocranialdysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia,craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentindysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia,encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,epithelial dysplasia, faciodigitogenital dysplasia, familial fibrousdysplasia of jaws, familial white folded dysplasia, fibromusculardysplasia, fibrous dysplasia of bone, florid osseous dysplasia,hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia,hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammarydysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondinidysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia,multiple epiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, ophthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be diagnosed, pro-nosed,prevented, and/or treated with fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to, benign dysproliferative disorders(e.g., benign tumors, fibrocystic conditions, tissue hypertrophy,intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia,keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solarkeratosis.

In another embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, maybe used to diagnose and/or pro-nose disorders associated with thetissue(s) in which the polypeptide of the invention is expressed.

In another embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the inventionconjugated to a toxin or a radioactive isotope, as described herein, maybe used to treat cancers and neoplasms, including, but not limited to,those described herein. In a further preferred embodiment, albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention conjugated to a toxin or a radioactiveisotope, as described herein, may be used to treat acute myelogenousleukemia.

Additionally, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may affect apoptosis,and therefore, would be useful in treating a number of diseasesassociated with increased cell survival or the inhibition of apoptosis.For example, diseases associated with increased cell survival or theinhibition of apoptosis that could be diagnosed, pro-nosed, prevented,and/or treated by polynucleotides, polypeptides, and/or agonists orantagonists of the invention, include cancers (such as follicularlymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection.

In preferred embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to inhibit growth, progression, and/or metastasis of cancers, inparticular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be diagnosed, prognosed, prevented, and/or treatedby fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, include, but are not limitedto, progression, and/or metastases of malignancies and related disorderssuch as leukemia (including acute leukemias (e.g., acute lymphocyticleukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be diagnosed,prognosed, prevented, and/or treated by fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, include AIDS; neurodegenerative disorders (such asAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,retinitis pigmentosa, cerebellar degeneration and brain tumor or priorassociated disease); autoimmune disorders (such as, multiple sclerosis,Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet'sdisease, Crohn's disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

Hyperproliferative diseases and/or disorders that could be diagnosed,prognosed, prevented, and/or treated by fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, include, but are not limited to, neoplasms located in theliver, abdomen, bone, breast, digestive system, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous system (central andperipheral), lymphatic system, pelvis, skin, soft tissue, spleen,thorax, and urogenital tract.

Similarly, other hyperproliferative disorders can also be diagnosed,prognosed, prevented, and/or treated by fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention. Examples of such hyperproliferative disorders include, butare not limited to: hypergammaglobulinemia, lymphoproliferativedisorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

Another preferred embodiment utilizes polynucleotides encoding albuminfusion proteins of the invention to inhibit aberrant cellular division,by gene therapy using the present invention, and/or protein fusions orfragments thereof.

Thus, the present invention provides a method for treating cellproliferative disorders by inserting into an abnormally proliferatingcell a polynucleotide encoding an albumin fusion protein of the presentinvention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating cell-proliferative disorders in individuals comprisingadministration of one or more active gene copies of the presentinvention to an abnormally proliferating cell or cells. In a preferredembodiment, polynucleotides of the present invention is a DNA constructcomprising a recombinant expression vector effective in expressing a DNAsequence encoding said polynucleotides. In another preferred embodimentof the present invention, the DNA construct encoding the fusion proteinof the present invention is inserted into cells to be treated utilizinga retrovirus, or more preferably an adenoviral vector (See G J. Nabel,et. al., PNAS 1999 96: 324-326, which is hereby incorporated byreference). In a most preferred embodiment, the viral vector isdefective and will not transform non-proliferating cells, onlyproliferating cells. Moreover, in a preferred embodiment, thepolynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDINA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

Moreover, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention of the presentinvention are useful in inhibiting the angiogenesis of proliferativecells or tissues, either alone, as a protein fusion, or in combinationwith other polypeptides directly or indirectly, as described elsewhereherein. In a most preferred embodiment, said anti-angiogenesis effectmay be achieved indirectly, for example, through the inhibition ofhematopoietic, tumor-specific cells, such as tumor-associatedmacrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53(1998), which is hereby incorporated by reference).

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be useful in inhibitingproliferative cells or tissues through the induction of apoptosis. Thesefusion proteins and/or polynucleotides may act either directly, orindirectly to induce apoptosis of proliferative cells and tissues, forexample in the activation of a death-domain receptor, such as tumornecrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-relatedapoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducingligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et al., Eur JBiochem 254(3):439-59 (1998), which is hereby incorporated byreference). Moreover, in another preferred embodiment of the presentinvention, these fusion proteins and/or polynucleotides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of these proteins, either alone or in combination with smallmolecule drubs or adjuviants, such as apoptonin, galectins,thioredoxins, anti-inflammatory proteins (See for example, Mutat Res400(1-2):447-55 (1998), Med Hypotheses. 50(5):423-33 (1998), Chem BiolInteract. Apr. 24;111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998),Int J Tissue React; 20(1):3-15 (1998), which are all hereby incorporatedby reference).

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention are useful in inhibiting themetastasis of proliferative cells or tissues. Inhibition may occur as adirect result of administering these albumin fusion proteins and/orpolynucleotides, or indirectly, such as activating the expression ofproteins known to inhibit metastasis, for example alpha 4 integrins,(See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is herebyincorporated by reference). Such thereapeutic affects of the presentinvention may be achieved either alone, or in combination with smallmolecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionto targeted cells expressing the a polypeptide bound by, that binds to,or associates with an albumin fuison protein of the invention. Albuminfusion proteins of the invention may be associated with withheterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions.

Albumin fusion proteins of the invention are useful in enhancing theimmunogenicity and/or antigenicity of proliferating cells or tissueseither directly, such as would occur if the albumin fusion proteins ofthe invention ‘vaccinated’ the immune response to respond toproliferative antigens and immunogens, or indirectly, such as inactivating the expression of proteins known to enhance the immuneresponse (e.g. chemokines), to said antigens and immunogens.

Renal Disorders

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, may be used to treat, prevent,diagnose, and/or prognose disorders of the renal system. Renal disorderswhich can be diagnosed, prognosed, prevented, and/or treated withcompositions of the invention include, but are not limited to, kidneyfailure, nephritis, blood vessel disorders of kidney, metabolic andcongenital kidney disorders, urinary disorders of the kidney, autoimmunedisorders, sclerosis and necrosis, electrolyte imbalance, and kidneycancers.

Kidney diseases which can be diagnosed, prognosed, prevented, and/ortreated with compositions of the invention include, but are not limitedto, acute kidney failure, chronic kidney failure, atheroembolic renalfailure, end-stage renal disease, inflammatory diseases of the kidney(e.g., acute glomerulonephritis, postinfectious glomerulonephritis,rapidly progressive glomerulonephritis, nephrotic syndrome, membranousglomerulonephritis, familial nephrotic syndrome, membranoproliferativeglomerulonephritis I and II, mesangial proliferative glomerulonephritis,chronic glomerulonephritis, acute tubulointerstitial nephritis, chronictubulointerstitial nephritis, acute post-streptococcalglomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronicnephritis, interstitial nephritis, and post-streptococcalglomerulonephritis), blood vessel disorders of the kidneys (e.g., kidneyinfarction, atheroembolic kidney disease, cortical necrosis, malignantnephrosclerosis, renal vein thrombosis, renal underperfusion, renalretinopathy, renal ischemia-reperfusion, renal artery embolism, andrenal artery stenosis), and kidney disorders-resulting form urinarytract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renallithiasis, nephrolithiasis), reflux nephropathy, urinary tractinfections, urinary retention, and acute or chronic unilateralobstructive uropathy.)

In addition, compositions of the invention can be used to diagnose,prognose, prevent, and/or treat metabolic and congenital disorders ofthe kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renaltubular acidosis, renal glycosuria, nephrogenic diabetes insipidus,cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renalrickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome,polycystic kidney disease, medullary cystic disease, medullary spongekidney, Alport's syndrome, nail-patella syndrome, congenital nephroticsyndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy,nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones,and membranous nephropathy), and autoimmune disorders of the kidney(e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgAnephropathy, and IgM mesangial proliferative glomerulonephritis).

Compositions of the invention can also be used to diagnose, prognose,prevent, and/or treat sclerotic or necrotic disorders of the kidney(e.g., glomerulosclerosis, diabetic nephropathy, focal segmentalglomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renalpapillary necrosis), cancers of the kidney (e.g., nephroma,hypernephroma, nephroblastoma, renal cell cancer, transitional cellcancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor),and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema,hydronephritis, proteinuria, hyponatremia, hype matremia, hypokalemia,hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, andhyperphosphatemia).

Compositions of the invention may be administered using any method knownin the art, including, but not limited to, direct needle injection atthe delivery site, intravenous injection, topical administration,catheter infusion, biolistic injectors, particle accelerators, gelfoamsponge depots, other commercially available depot materials, osmoticpumps, oral or suppositorial solid pharmaceutical formulations,decanting or topical applications during surgery, aerosol delivery. Suchmethods are known in the art. Compositions of the invention may beadministered as part of a Therapeutic, described in more detail below.Methods of delivering polynucleotides of the invention are described inmore detail herein.

Cardiovascular Disorders

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, may be used to treat, prevent,diagnose, and/or prognose cardiovascular disorders, including, but notlimited to, peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include, but are not limited to, cardiovascularabnormalities, such as arterio-arterial fistula, arteriovenous fistula,cerebral arteriovenous malformations, congenital heart defects,pulmonary atresia, and Scimitar Syndrome. Congenital heart defectsinclude, but are not limited to, aortic coarctation, cor triatriatum,coronary vessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulmonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

Cardiovascular disorders also include, but are not limited to, heartdisease, such as arrhythmias, carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure,congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, hearthypertrophy, congestive cardiomyopathy, left ventricular hypertrophy;right ventricular hypertrophy, post-infarction heart rupture,ventricular septal rupture, heart valve diseases, myocardial diseases,myocardial ischemia, pericardial effusion, pericarditis (includingconstrictive and tuberculous), pneumopericardium, postpericardiotomysyndrome, pulmonary heart disease, rheumatic heart disease, ventriculardysfunction, hyperemia, cardiovascular pregnancy complications, ScimitarSyndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include, but are not limited to, sinus arrhythmia, atrialfibrillation, atrial flutter, bradycardia, extrasystole, Adams-StokesSyndrome, bundle-branch block, sinoatrial block, long QT syndrome,parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitationsyndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome,tachycardias, and ventricular fibrillation. Tachycardias includeparoxysmal tachycardia, supraventricular tachycardia, acceleratedidioventricular rhythm, atrioventricular nodal reentry tachycardia,ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrialnodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, andventricular tachycardia.

Heart valve diseases include, but are not limited to, aortic valveinsufficiency, aortic valve stenosis, hear murmurs, aortic valveprolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valveinsufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valveinsufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspidvalve insufficiency, and tricuspid valve stenosis. Myocardial diseasesinclude, but are not limited to, alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include, but are not limited to, coronary disease,such as angina pectoris, coronary aneurysm, coronary arteriosclerosis,coronary thrombosis, coronary vasospasm, myocardial infarction andmyocardial stunning Cardiovascular diseases also include vasculardiseases such as aneurysms, angiodysplasia, angiomatosis, bacillaryangiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,Sturge-Weber Syndrome, angrioneurotic edema, aortic diseases, Takayasu'sArteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases,arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders,diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include, but are not limited to, dissecting aneurysms, falseaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms,cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliacaneurysms.

Arterial occlusive diseases include, but are not limited to,arteriosclerosis, intermittent claudication, carotid stenosis,fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoyadisease, renal artery obstruction, retinal artery occlusion, andthromboanagiitis obliterans.

Cerebrovascular disorders include, but are not limited to, carotidartery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis. Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

Embolisms include, but are not limited to, air embolisms, amniotic fluidembolisms, cholesterol embolisms, blue toe syndrome, fat embolisms,pulmonary embolisms, and thromoboembolisms. Thrombosis include, but arenot limited to, coronary thrombosis, hepatic vein thrombosis, retinalvein occlusion, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, and thrombophlebitis.

Ischemic disorders include, but are not limited to, cerebral ischemia,ischemic colitis, compartment syndromes, anterior compartment syndrome,myocardial ischemia, reperfusion injures, and peripheral limb, ischemia.Vasculitis includes, but is not limited to, aortitis, arteritis,Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph nodesyndrome, thromboanguitis obliterans, hypersensitivity vasculitis,Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener'sgranulomatosis.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be administered using anymethod known in the art, including, but not limited to, direct needleinjection at the delivery site, intravenous injection, topicaladministration, catheter infusion, biolistic injectors, particleaccelerators, gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps, oral or suppositorial solid pharmaceuticalformulations, decanting or topical applications during surgery, aerosoldelivery. Such methods are known in the art. Methods of deliveringpolynucleotides are described in more detail herein.

Respiratory Disorders

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be used to treat, prevent,diagnose, and/or prognose diseases and/or disorders of the respiratorysystem.

Diseases and disorders of the respiratory system include, but are notlimited to, nasal vestibulitis, nonallergic rhinitis (e.g., acuterhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis),nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the noseand juvenile papillomas, vocal cord polyps, nodules (singer's nodules),contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g.,viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngealabscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer ofthe nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g.,squamous cell carcinoma, small cell (oat cell) carcinoma, large cellcarcinoma, and adenocarcinoma), allergic disorders (eosinophilicpneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergicalveolitis, allergic interstitial pneumonitis, organic dustpneumoconiosis, allergic bronchopulmonary aspergillosis, asthma,Wegener's granulomatosis (granulomatous vasculitis), Goodpasture'ssyndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcuspneumoniae. (pneumoncoccal pneumonia), Staphylococcus aureus(staphylococcal pneumonia), Gram-negative bacterial pneumonia (causedby, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniaepneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila(Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), andviral pneumonia (e.g., influenza, chickenpox (varicella).

Additional diseases and disorders of the respiratory system include, butare not limited to bronchiolitis, polio (poliomyelitis), croup,respiratory syncytial viral infection, mumps, erythema infectiosum(fifth disease), roseola infantum, progressive rubella panencephalitis,german measles, and subacute sclerosing panencephalitis), fungalpneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis,fungal infections in people with severely suppressed immune systems(e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis,caused by Aspergillus spp.; candidiasis, caused by Candida; andmucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypicalpneumonias (e.g., Mycoplasma and Chlamydia spp.), opportunisticinfection pneumonia, nosocomial pneumonia, chemical pneumonitis, andaspiration pneumonia, pleural disorders (e.g., pleurisy, pleuraleffusion, and pneumothorax (e.g., simple spontaneous pneumothorax,complicated spontaneous pneumothorax, tension pneumothorax)),obstructive airway diseases (e.g., asthma, chronic obstructive pulmonarydisease (COPD), emphysema, chronic or acute bronchitis), occupationallung diseases (erg., silicosis, black lung (coal workers'pneumoconiosis), asbestosis, berylliosis, occupational asthsma,byssinosis, and benign pneumoconioses), Infiltrative Lung Disease (e.g.,pulmonary fibrosis (e.g., fibrosing alveolitis, usual interstitialpneumonia), idiopathic pulmonary fibrosis, desquarnative interstitialpneumonia, lymphoid interstitial pneumonia, histiocytosis X (e.g.,Letterer-Siwe disease, Hand-Schüller-Christian disease, eosinophilicgranuloma), idiopathic pulmonary hemosiderosis, sarcoidosis andpulmonary alveolar proteinosis), Acute respiratory distress syndrome(also called, e.g., adult respiratory distress syndrome), edema,pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis,atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus orLegionella pneumophila), and cystic fibrosis.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-11 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention. Malignant and metastatic conditions which can betreated with the polynucleotides and polypeptides, or agonists orantagonists of the invention include, but are not limited to,malignancies, solid tumors, and cancers described herein and otherwiseknown in the art (for a review of such disorders, see Fishman et al.,Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, thepresent invention provides a method of treating an angiogenesis-relateddisease and/or disorder, comprising administering to an individual inneed thereof a therapeutically effective amount of an albumin fusionprotein of the invention and/or polynucleotides encoding an albuminfusion protein of the invention. For example, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be utilized in a variety of additional methods in order totherapeutically treat a cancer or tumor. Cancers which may be treatedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include, but are not limited tosolid tumors, including prostate, lung, breast, ovarian, stomach,pancreas, larynx, esophagus, testes, liver, parotid, biliary tract,colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroidcancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi'ssarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer;advanced malignancies; and blood born tumors such as leukemias. Forexample, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be deliveredtopically, in order to treat cancers such as skin cancer, head and necktumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beutilized to treat superficial forms of bladder cancer by, for example,intravesical administration. Albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be delivered directly into the tumor, or near the tumor site, viainjection or a catheter. Of course, as the artisan of ordinary skillwill appreciate, the appropriate mode of administration will varyaccording to the cancer to be treated. Other modes of delivery arediscussed herein.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be useful in treating otherdisorders, besides cancers, which involve angiogenesis. These disordersinclude, but are not limited to: benign tumors, for example hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;artheroscleric plaques; ocular angiogenic diseases, for example,diabetic retinopathy, retinopathy of prematurity, macular degeneration,corneal graft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vesselgrowth) of the eye; rheumatoid arthritis; psoriasis; delayed woundhealing; endometriosis; vasculogenesis; granulations; hypertrophic scars(keloids); nonunion fractures; scleroderma; trachoma; vascularadhesions; myocardial angiogenesis; coronary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; fibromuscular dysplasia; woundgranulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods areprovided for treating hypertrophic scars and keloids, comprising thestep of administering albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention to ahypertrophic scar or keloid.

Within one embodiment of the present invention fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are directly injected into a hypertrophic scar or keloid, inorder to prevent the progression of these lesions. This therapy is ofparticular value in the prophylactic treatment of conditions which areknown to result in the development of hypertrophic scars and keloids(e.g., burns), and is preferably initiated after the proliferative phasehas had time to progress (approximately 14 days after the initialinjury), but before hypertrophic scar or keloid development. As notedabove, the present invention also provides methods for treatingneovascular diseases of the eye, including for example, cornealneovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroplasia and macular degeneration.

Moreover, Ocular disorders associated with neovascularization which canbe treated with the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to: neovascular glaucoma, diabeticretinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

Thus, within one aspect of the present invention methods are providedfor treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (e.g., fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention) to the cornea, such that the formation of blood vessels isinhibited. Briefly, the cornea is a tissue which normally lacks-bloodvessels. In certain pathological conditions however, capillaries mayextend into the cornea from the pericorneal vascular plexus of thelimbus. When the cornea becomes vascularized, it also becomes clouded,resulting in a decline in the patient's visual acuity. Visual loss maybecome complete if the cornea completely opacitates. A wide variety ofdisorders can result in corneal neovascularization, including forexample, corneal infections (e.g., trachoma, herpes simplex keratitis,leishmaniasis and onchocerciasis), immunological processes (e.g., graftrejection and Stevens-Johnson's syndrome), alkali burns, trauma,inflammation (of any cause), toxic and nutritional deficiency states andas a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may beprepared for topical administration in saline (combined with any of thepreservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

Within other embodiments, the compounds described above may be injecteddirectly into the corneal stroma by an ophthalmologist under microscopicguidance. The preferred site of injection may vary with the morphologyof the individual lesion, but the goal of the administration would be toplace the composition at the advancing front of the vasculature (i.e.,interspersed between the blood vessels and the normal cornea). In mostcases this would involve perilimbic corneal injection to “protect” thecornea from the advancing blood vessels. This method may also beutilized shortly after a corneal insult in order to prophylacticallyprevent corneal neovascularization. In this situation the material couldbe injected in the perilimbic cornea interspersed between the corneallesion and its undesired potential limbic blood supply. Such methods mayalso be utilized in a similar fashion to prevent capillary invasion oftransplanted corneas. In a sustained-release form injections might onlybe required 2-3 times per year. A steroid could also be added to theinjection solution to reduce inflammation resulting from the injectionitself.

Within another aspect of the present invention, methods are provided fortreating neovascular glaucoma, comprising the step of administering to apatient a therapeutically effective amount of an albumin fusion proteinof the invention and/or polynucleotides encoding an albumin fusionprotein of the invention to the eye, such that the formation of bloodvessels is inhibited. In one embodiment, the compound may beadministered topically to the eye in order to treat early forms ofneovascular glaucoma. Within other embodiments, the compound may beimplanted by injection into the region of the anterior chamber angle.Within other embodiments, the compound may also be placed in anylocation such that the compound is continuously released into theaqueous humor. Within another aspect of the present invention, methodsare provided for treating proliferative diabetic retinopathy, comprisingthe step of administering to a patient a therapeutically effectiveamount of an albumin fusion protein of the invention and/orpolynucleotides encoding an albumin fusion protein of the invention tothe eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided fortreating retrolental fibroplasia, comprising the step of administeringto a patient a therapeutically effective amount of an albumin fusionprotein of the invention and/or polynucleotides encoding an albuminfusion protein of the invention to the eye, such that the formation ofblood vessels is inhibited. The compound may be administered topically,via intravitreous injection and/or via intraocular implants.

Additionally, disorders which can be treated with fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention include, but are not limited to, hemangioma, arthritis,psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing,granulations, hemophilic joints, hypertrophic scars, nonunion fractures,Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, andvascular adhesions.

Moreover, disorders and/or states, which can be treated, prevented,diagnosed, and/or pro-nosed with the the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention of the invention include, but are not limited to, solidtumors, blood born tumors such as leukemias, tumor metastasis, Kaposi'ssarcoma, benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis,psoriasis, ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, and uvietis, delayed wound healing,endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compoundsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method. Albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention may also be used in controllingmenstruation or administered as either a peritoneal lavage fluid or forperitoneal implantation in the treatment of endometriosis.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be incorporated intosurgical sutures in order to prevent stitch granulomas.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may be utilized in a widevariety of surgical procedures. For example, within one aspect of thepresent invention a compositions (in the form of, for example, a sprayor film) may be utilized to coat or spray an area prior to removal of atumor, in order to isolate normal surrounding tissues from malignanttissue, and/or to prevent the spread of disease to surrounding tissues.Within other aspects of the present invention, compositions (e.g., inthe form of a spray) may be delivered via endoscopic procedures in orderto coat tumors, or inhibit angiogenesis in a desired locale. Within yetother aspects of the present invention, surgical meshes which have beencoated with anti-angiogenic compositions of the present invention may beutilized in any procedure wherein a surgical mesh might be utilized. Forexample, within one embodiment of the invention a surgical mesh ladenwith an anti-angiogenic composition may be utilized during abdominalcancer resection surgery (e.g., subsequent to colon resection) in orderto provide support to the structure, and to release an amount of theanti-angiogenic factor.

Within further aspects of the present invention, methods are providedfor treating tumor excision sites, comprising administering albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention to the resection margins of a tumorsubsequent to excision, such that the local recurrence of cancer and theformation of new blood vessels at the site is inhibited. Within oneembodiment of the invention, the anti-angiogenic compound isadministered directly to the tumor excision site (e.g., applied byswabbing, brushing or otherwise coating the resection mar-ins of thetumor with the anti-angiogenic compound). Alternatively, theanti-angiogenic compounds may be incorporated into known surgical pastesprior to administration. Within particularly preferred embodiments ofthe invention, the anti-angiogenic compounds are applied after hepaticresections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be administered to the resection margin of a wide varietyof tumors, including for example, breast, colon, brain and hepatictumors. For example, within one embodiment of the invention,anti-angiogenic compounds may be administered to the site of aneurological tumor subsequent to excision, such that the formation ofnew blood vessels at the site are inhibited.

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may also beadministered along with other anti-angiogenic factors. Representativeexamples of other anti-angiogenic factors include: Anti-Invasive Factor,retinoic acid and derivatives thereof, paclitaxel, Suramin, TissueInhibitor of Metalloproteinase-1, Tissue Inhibitor ofMetalloproteinase-2, Plasminogen Activator Inhibitor-1, PlasminogenActivator Inhibitor-2, and various forms of the lighter “d group”transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium; and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, (1992)); Chymostatin. (Tomkinson et al.,Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, (1987)); Bisantrene(National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4chloroanthronilic acid disodium or “CCA”; Takeuchi et al., AgentsActions 36:312-316, (1992)); Thalidomide; Angostatic steroid; AGM-1470;carboxynaininolmidazole; and metalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated, prevented, diagnosed, and/or prognosedusing fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, include cancers (such asfollicular lymphomas, carcinomas with p53 mutations, andhormone-dependent tumors, including, but not limited to colon cancer,cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection.

In preferred embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to inhibit growth, progression, and/or metasis of cancers, inparticular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated,prevented, diagnosed, and/or prognesed using fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention, include, but are not limited to, AIDS; neurodegenerativedisorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration andbrain tumor or prior associated disease); autoimmune disorders (such as,multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.host disease, ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury (e.g.,hepatitis related liver injury, ischemia/reperfusion injury, cholestosis(bile duct injury) and liver cancer); toxin-induced liver disease (suchas that caused by alcohol), septic shock, cachexia and anorexia. WoundHealing and Epithelial Cell Proliferation In accordance with yet afurther aspect of the present invention, there is provided a process forutilizing fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, for therapeuticpurposes, for example, to stimulate epithelial cell proliferation andbasal keratinocytes for the purpose of wound healing, and to stimulatehair follicle production and healing of dermal wounds. Albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention, may be clinically useful in stimulating woundhealing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as urermia, malnutrition, vitamin deficiencies andcomplications associated with systemic treatment with steroids,radiation therapy and antineoplastic drugs and antimetabolites. Albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention, could be used to promote dermalreestablishment subsequent to dermal loss

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, could be used to increase theadherence of skin grafts to a wound bed and to stimulatere-epithelialization from the wound bed. The following are types ofgrafts that fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could be used toincrease adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. Albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention, can be used to promote skin strength and toimprove the appearance of aced skin.

It is believed that fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, willalso produce chances in hepatocyte proliferation, and epithelial cellproliferation in the lung, breast, pancreas, stomach, small intestine,and large intestine. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, couldpromote proliferation of epithelial cells such as sebocytes, hairfollicles, hepatocytes, type II pneumocytes, mucin-producing gobletcells, and other epithelial cells and their progenitors contained withinthe skin, lung, liver, and gastrointestinal tract. Albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, could also be used to reducethe side effects of gut toxicity that result from radiation,chemotherapy treatments or viral infections. Albumin fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention, may have a cytoprotective effect on the small intestinemucosa. Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may also stimulatehealing of mucositis (mouth ulcers) that result from chemotherapy andviral infections.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, could further be used in fullregeneration of skin in full and partial thickness skin defects,including burns, (i.e., repopulation of hair follicles, sweat glands,and sebaceous glands), treatment of other skin defects such aspsoriasis. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, couldbe used to treat epidermolysis bullosa, a defect in adherence of theepidermis to the underlying dermis which results in frequent, open andpainful blisters by accelerating reepithelialization of these lesions.Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, could also be used to treatgastric and doudenal ulcers and help heal by scar formation of themucosal lining and regeneration of glandular mucosa and duodenal mucosallining more rapidly. Inflammatory bowel diseases, such as Crohn'sdisease and ulcerative colitis, are diseases which result in destructionof the mucosal surface of the small or large intestine, respectively.Thus, fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, could be used to promote theresurfacing of the mucosal surface to aid more rapid healing and toprevent progression of inflammatory bowel disease. Treatment with fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention, is expected to have a significant effect onthe production of mucus throughout the gastrointestinal tract and couldbe used to protect the intestinal mucosa from injurious substances thatare ingested or following surgery. Albumin fusion proteins of theinvention, and/or polynucleotides encoding albumin fusion proteins ofthe invention, could be used to treat diseases associate with the underexpression.

Moreover, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could be used toprevent and heal damage to the lungs due to various pathological states.Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, which could stimulateproliferation and differentiation and promote the repair of alveoli andbrochiolar epithelium to prevent or treat acute or chronic lung damage.For example, emphysema, which results in the progressive loss of aveoli,and inhalation injuries, i.e., resulting from smoke inhalation andburns, that cause necrosis of the bronchiolar epithelium and alveolicould be effectively treated using polynucleotides or polypeptides,agonists or antagonists of the present invention. Also fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention, could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, could stimulate theproliferation and differentiation of hepatocytes and, thus, could beused to alleviate or treat liver diseases and pathologies such asfulminant liver failure caused by cirrhosis, liver damage caused byviral hepatitis and toxic substances (i.e., acetaminophen, carbontetraholoride and other hepatotoxins known in the art).

In addition, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could be used treator prevent the onset of diabetes mellitus. In patients with newlydiagnosed Types I and II diabetes, where some islet cell functionremains, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could be used tomaintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention, could be used as an auxiliary in islet cell transplantationto improve or promote islet cell function.

Neural Activity and Neurological Diseases

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be used for thediagnosis and/or treatment of diseases, disorders, damage or injury ofthe brain and/or nervous system. Nervous system disorders that can betreated with the compositions of the invention (e.g., fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention), include, but are not limited to, nervous systeminjuries, and diseases or disorders which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated in a patient(including human and non-human mammalian patients) according to themethods of the invention, include but are not limited to, the followinglesions of either the central (including spinal cord, brain) orperipheral nervous systems: (1) ischemic lesions, in which a lack ofoxygen in a portion of the nervous system results in neuronal injury ordeath, including cerebral infarction or ischemia, or spinal cordinfarction or ischemia; (2) traumatic lesions, including lesions causedby physical injury or associated with surgery, for example, lesionswhich sever a portion of the nervous system, or compression injuries;(3) malignant lesions, in which a portion of the nervous system isdestroyed or injured by malignant tissue which is either a nervoussystem associated malignancy or a malignancy derived from non-nervoussystem tissue; (4) infectious lesions, in which a portion of the nervoussystem is destroyed or injured as a result of infection, for example, byan abscess or associated with infection by human immunodeficiency virus,herpes zoster, or herpes simplex virus or with Lyme disease,tuberculosis, or syphilis; (5) degenerative lesions, in which a portionof the nervous system is destroyed or injured as a result of adegenerative process including but not limited to, degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases or disorders, in which a portion of thenervous system is destroyed or injured by a nutritional disorder ordisorder of metabolism including, but not limited to, vitamin B12deficiency, folic acid deficiency, Wemicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

In one embodiment, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to protect neural cells from the damaging effects of hypoxia. In afurther preferred embodiment, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to protect neural cells from the damaging effects ofcerebral hypoxia. According to this embodiment, the compositions of theinvention are used to treat or prevent neural cell injury associatedwith cerebral hypoxia. In one non-exclusive aspect of this embodiment,the albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, are used to treat orprevent neural cell injury associated with cerebral ischemia. In anothernon-exclusive aspect of this embodiment, the albumin fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention are used to treat or prevent neural cell injury associatedwith cerebral infarction.

In another preferred embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to treat or prevent neural cell injury associatedwith a stroke. In a specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to treat or prevent cerebral neural cell injuryassociated with a stroke.

In another preferred embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to treat or prevent neural cell injury associatedwith a heart attack. In a specific embodiment, albumin fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention are used to treat or prevent cerebral neural cellinjury associated with a heart attack.

The compositions of the invention which are useful for treating orpreventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture either in the presence or absence of hypoxia or hypoxicconditions; (2) increased sprouting of neurons in culture or in vivo;(3) increased production of a neuron-associated molecule in culture orin vivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, in Zhang et al., ProcNatl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci.,10:3507-15 (1990); increased sprouting of neurons may be detected bymethods known in the art, such as, for example, the methods set forth inPestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann.Rev. Neurosci., 4:17-42 (1981); increased production ofneuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., using techniquesknown in the art and depending on the molecule to be measured; and motorneuron dysfunction may be measured by assessing the physicalmanifestation of motor neuron disorder, e.g., weakness, motor neuronconduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treatedaccording to the invention include, but are not limited to, disorderssuch as infarction, infection, exposure to toxin, trauma, surgicaldamage, degenerative disease or malignancy that may affect motor neuronsas well as other components of the nervous system, as well as disordersthat selectively affect neurons such as amyotrophic lateral sclerosis,and including, but not limited to, progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Further, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may play a role inneuronal survival; synapse formation; conductance; neuraldifferentiation, etc. Thus, compositions of the invention (includingfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention) may be used to diagnose and/or treator prevent diseases or disorders associated with these roles, including,but not limited to, learning and/or cognition disorders. Thecompositions of the invention may also be useful in the treatment orprevention of neurodegenerative disease states and/or behaviouraldisorders. Such neurodegenerative disease states and/or behavioraldisorders include, but are not limited to, Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, Tourette Syndrome,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,panic disorder, learning disabilities, ALS, psychoses, autism, andaltered behaviors, including disorders in feeding, sleep patterns,balance, and perception. In addition, compositions of the invention mayalso play a role in the treatment, prevention and/or detection ofdevelopmental disorders associated with the developing embryo, orsexually-linked disorders.

Additionally, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may be useful inprotecting neural cells from diseases, damage, disorders, or injury,associated with cerebrovascular disorders including, but not limited to,carotid artery diseases (e.g., carotid artery thrombosis, carotidstenosis, or Moyamoya Disease), cerebral amyloid angiopathy, cerebralaneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformations, cerebral artery diseases, cerebral embolismand thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, orWallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subduralhematoma, or subarachnoid hemorrhage), cerebral infarction, cerebralischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome,or vertebrobasilar insufficiency), vascular dementia (e.g.,multi-infarct), leukomalacia, periventricular, and vascular headache(e.g., cluster headache or migraines).

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, for therapeutic purposes, for example, to stimulateneurological cell proliferation and/or differentiation. Therefore,fusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention may be used to treat and/or detectneurologic diseases. Moreover, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, canbe used as a marker or detector of a particular nervous system diseaseor disorder.

Examples of neurologic diseases which can be treated or detected withfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include, brain diseases, such asmetabolic brain diseases which includes phenylketonuria such as maternalphenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenasecomplex deficiency, Wernicke's Encephalopathy, brain edema, brainneoplasms such as cerebellar neoplasms which include infratentorialneoplasms, cerebral ventricle neoplasms such as choroid plexusneoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavandisease, cerebellar diseases such as cerebellar ataxia which includespinocerebellar degeneration such as ataxia telangiectasia, cerebellardyssynergia, Friederich's Ataxia, Machado-Joseph Disease,olivopontocerebellar atrophy, cerebellar neoplasms such asinfratentorial neoplasms, diffuse cerebral sclerosis such asencephalitis periaxialis, globoid cell leukodystrophy, metachromaticleukodystrophy and subacute sclerosing panencephalitis.

Additional neurologic diseases which can be treated or detected withfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include cerebrovascular disorders (suchas carotid artery diseases which include carotid artery thrombosis,carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy,cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformations, cerebral artery diseases, cerebral embolismand thrombosis such as carotid artery thrombosis, sinus thrombosis andWallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma,subdural hematoma and subarachnoid hemorrhage, cerebral infarction,cerebral ischemia such as transient cerebral ischemnia, Subclavian StealSyndrome and vertebrobasilar insufficiency, vascular dementia such asmulti-infarct dementia, periventricular leukomalacia, vascular headachesuch as cluster headache and migraine.

Additional neurologic diseases which can be treated or detected withfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include dementia such as AIDS DementiaComplex, presenile dementia such as Alzheimer's Disease andCreutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Diseaseand progressive supranuclear palsy, vascular dementia such asmulti-infarct dementia, encephalitis which include encephalitisperiaxialis, viral encephalitis such as epidemic encephalitis, JapaneseEncephalitis, St. Louis Encephalitis, tick-borne encephalitis and WestNile Fever, acute disseminated encephalomyelitis, meningoencephalitissuch as uveomeningoencephalitic syndrome, Postencephalitic ParkinsonDisease and subacute sclerosing panencephalitis, encephalomalacia suchas periventricular leukomalacia, epilepsy such as generalized epilepsywhich includes infantile spasms, absence epilepsy, myoclonic epilepsywhich includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsysuch as complex partial epilepsy, frontal lobe epilepsy and temporallobe epilepsy, post-traumatic epilepsy, status epilepticus such asEpilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.

Additional neurologic diseases which can be treated or detected withfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include hydrocephalus such asDandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamicdiseases such as hypothalamic neoplasms, cerebral malaria, narcolepsywhich includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor,Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebraltoxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, centralnervous system infections such as AIDS Dementia Complex, Brain Abscess,subdural empyema, encephalomyelitis such as Equine Encephalomyelitis,Venezuelan Equine Encephalomyelitis, Necrotizing HemorrhagicEncephalomyelitis, Visna, and cerebral malaria.

Additional neurologic diseases which can be treated or detected withfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include meningitis such asarachnoiditis, aseptic meningtitis such as viral meningtitis whichincludes lymphocytic choriomeningitis, Bacterial meningtitis whichincludes Haemophilus Meningtitis, Listeria Meningtitis, MeningococcalMeningtitis such as Waterhouse-Friderichsen Syndrome, PneumococcalMeningtitis and meningeal tuberculosis, fungal meningitis such asCryptococcal Meningtitis, subdural effusion, meningoencephalitis such asuvemeningoencephalitic syndrome, myelitis such as transverse myelitis,neurosyphilis such as tabes dorsalis, poliomyelitis which includesbulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases(such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy,Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebraltoxoplasmosis.

Additional neurologic diseases which can be treated or detected withfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include central nervous systemneoplasms such as brain neoplasms that include cerebellar neoplasms suchas infratentorial neoplasms, cerebral ventricle neoplasms such aschoroid plexus neoplasms, hypothalamic neoplasms and supratentorialneoplasms, meningeal neoplasms, spinal cord neoplasms which includeepidural neoplasms, demyelinating diseases such as Canavan Diseases,diffuse cerebral sceloris which includes adrenoleukodystrophy,encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebralsclerosis such as metachromatic leukodystrophy, allergicencephalomyelitis, necrotizing hemorrhagic encephalomyelitis,progressive multifocal leukoencephalopathy, multiple sclerosis, centralpontine myelinolysis, transverse myelitis, neuromyelitis optica,Scrapie. Swayback, Chronic Fatigue Syndrome, Visna, High PressureNervous Syndrome, vlieningism, spinal cord diseases such as amyotoniacongenita, amyotrophic lateral sclerosis, spinal muscular atrophy suchas Werdnig-Hoffmann Disease, spinal cord compression, spinal cordneoplasms such as epidural neoplasms, syringomyelia, Tabes Dorsalis,Stiff-Man Syndrome, mental retardation such as Angelman Syndrome,Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidosessuch as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease,Hartnup Disease, homocystinuria, Laurence-Moon-Biedl Syndrome,Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such asfucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal syndrome,phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome,Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGRSyndrome, nervous system abnormalities such as holoprosencephaly, neuraltube defects such as anencephaly which includes hydrangencephaly,Arnold-Chairi Deformity, encephalocele, meningocele, meningomyelocele,spinal dysraphism such as spina bifida cystica and spina bifida occulta.

Additional neurologic diseases which can be treated or detected withfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include hereditary motor and sensoryneuropathies which include Charcot-Marie Disease, Hereditary opticatrophy, Refsum's Disease, hereditary spastic paraplegia,Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathiessuch as Congenital Analgesia and Familial Dysautonomia, Neurologicmanifestations (such as agnosia that include Gerstmann's Syndrome,Amnesia such as retrograde amnesia, apraxia, neurogenic bladder,cataplexy, communicative disorders such as hearing disorders thatincludes deafness, partial hearing loss, loudness recruitment andtinnitus, language disorders such as aphasia which include agraphia,anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as AcquiredDyslexia, language development, disorders, speech disorders such asaphasia which includes anomia, broca aphasia and Wemicke Aphasia,articulation disorders, communicative disorders such as speech disorderswhich include dysarthria, echolalia, mutism and stuttering, voicedisorders such as aphonia and hoarseness, decerebrate state, delirium,fasciculation, hallucinations, meningism, movement disorders such asangelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia,muscle hypotonia, myoclonus, tic, torticollis and tremor, musclehypertonia such as muscle rigidity such as stiff-man syndrome, musclespasticity, paralysis such as facial paralysis which includes HerpesZoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such asdiplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressiveexternal ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis,Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome,quadriplegia, respiratory paralysis and vocal cord paralysis, paresis,phantom limb, taste disorders such as ageusia and dysgeusia, visiondisorders such as amblyopia, blindness, color vision defects, diplopia,hemianopsia, scotoma and subnormal vision, sleep disorders such ashypersomnia which includes Kleine-Levin Syndrome, insomnia, andsomnambulism, spasm such as trismus, unconsciousness such as coma,persistent vegetative state and syncope and vertigo, neuromusculardiseases such as amyotonia congenita, amyotrophic lateral sclerosis,Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscularatrophy such as spinal muscular atrophy, Charcot-Marie Disease andWerdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, MluscularDystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita,Nemaline Myopathy, Familial Periodic Paralysis, M ultiplexParamyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome,peripheral nervous system diseases such as acrodynia, amyloidneuropathies, autonomic nervous system diseases such as Adie's Syndrome,Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, ReflexSympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseasessuch as Acoustic Nerve Diseases such as Acoustic Neuroma which includesNeurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includesamblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia suchas Duane's Syndrome, Horner's Syndrome, Chronic Progressive ExternalOphthalmoplegia which includes Kearns Syndrome, Strabismus such asEsotropia and Exotropia, Oculomotor Nerve Paralysis, Optic NerveDiseases such as Optic Atrophy which includes Hereditary Optic Atrophy,Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica,Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, DemyelinatingDiseases such as Neuromyelitis Optica and Swayback, and Diabeticneuropathies such as diabetic foot.

Additional neurologic diseases which can be treated or detected withfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include nerve compression syndromessuch as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outletsyndrome such as cervical rib syndrome, ulnar nerve compressionsyndrome, neuralgia such as causalgia, cervico-brachial neuralgia,facial neuralgia and trigeminal neuralgia, neuritis such as experimentalallergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritisand radiculities such as polyradiculitis, hereditary motor and sensoryneuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy,Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-HoffmannDisease, Hereditary Sensory and Autonomic Neuropathies which includeCongenital Analgesia and Familial Dysautonomia, POEMS Syndrome,Sciatica, Gustatory Sweating and Tetany).

Endocrine Disorders

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, may be used to treat, prevent,diagnose, and/or prognose disorders and/or diseases related to hormoneimbalance, and/or disorders or diseases of the endocrine system.

Hormones secreted by the glands of the endocrine system control physicalgrowth, sexual function, metabolism, and other functions. Disorders maybe classified in two ways: disturbances in the production of hormones,and the inability of tissues to respond to hormones. The etiology ofthese hormone imbalance or endocrine system diseases, disorders orconditions may be genetic, somatic, such as cancer and some autoimmunediseases, acquired (e.g., by chemotherapy, injury or toxins), orinfectious. Moreover, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention can beused as a marker or detector of a particular disease or disorder relatedto the endocrine system and/or hormone imbalance.

Endocrine system and/or hormone imbalance and/or diseases encompassdisorders of uterine motility including, but not limited to:complications with pregnancy and labor (e.g., pre-term labor, post-termpregnancy, spontaneous abortion, and slow or stopped labor); anddisorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea andendometriosis).

Endocrine system and/or hormone imbalance disorders and/or diseasesinclude disorders and/or diseases of the pancreas, such as, for example,diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis,pheochromocytoma-islet cell tumor syndrome; disorders and/or diseases ofthe adrenal glands such as, for example, Addison's Disease,corticosteroid deficiency, virilizing disease, hirsutism, Cushing'sSyndrome, hyperaldosteronism, pheochromocytoma; disorders and/ordiseases of the pituitary gland, such as, for example, hyperpituitarism,hypopituitarism, pituitary dwarfism, pituitary adenoma,panhypopituitarism, acromegaly, gigantism; disorders and/or diseases ofthe thyroid, including but not limited to, hyperthyroidism,hypothyroidism, Plummer's disease, Graves' disease (toxic diffusegoiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis,subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis),Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormonecoupling defect, thymic aplasia, Hurthle cell tumours of the thyroid,thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma;disorders and/or diseases of the parathyroid, such as, for example,hyperparathyroidism, hypoparathyroidism; disorders and/or diseases ofthe hypothalamus.

In addition, endocrine system and/or hormone imbalance disorders and/ordiseases may also include disorders and/or diseases of the testes orovaries, including cancer. Other disorders and/or diseases of the testesor ovaries further include, for example, ovarian cancer, polycysticovary syndrome, Klinefelter's syndrome, vanishing testes syndrome(bilateral anorchia), congenital absence of Leydig's cells,cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillaryhaemangioma of the testis (benign), neoplasias of the testis andneo-testis.

Moreover, endocrine system and/or hormone imbalance disorders and/ordiseases may also include disorders and/or diseases such as, forexample, polyglandular deficiency syndromes, pheochromocytoma,neuroblastoma, multiple Endocrine neoplasia, and disorders and/orcancers of endocrine tissues.

In another embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, maybe used to diagnose, pro-nose, prevent, and/or treat endocrine diseasesand/or disorders associated with the tissue(s) in which the Therapeuticprotein corresponding to the Therapeutic protein portion of the albuminprotein of the invention is expressed,

Reproductive System Disorders

The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be used for thediagnosis, treatment, or prevention of diseases and/or disorders of thereproductive system. Reproductive system disorders that can be treatedby the compositions of the invention, include, but are not limited to,reproductive system injuries, infections, neoplastic disorders,congenital defects, and diseases or disorders which result ininfertility, complications with pregnancy, labor, or parturition, andpostpartum difficulties.

Reproductive system disorders and/or diseases include diseases and/ordisorders of the testes, including testicular atrophy, testicularfeminization, cryptorchism (unilateral and bilateral), anorchia, ectopictestis, epididymitis and orchitis (typically resulting from infectionssuch as, for example, gonorrhea, mumps, tuberculosis, and syphilis),testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas,embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sactumors, and teratomas), stromal tumors (e.g., Leydig cell tumors),hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, anddisorders of sperm production (e.g., immotile cilia syndrome, aspermia,asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).

Reproductive system disorders also include disorders of the prostategland, such as acute non-bacterial prostatitis, chronic non-bacterialprostatitis, acute bacterial prostatitis, chronic bacterial prostatitis,prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia,benign prostatic hypertrophy or hyperplasia, and prostate neoplasticdisorders, including adenocarcinomas, transitional cell carcinomas,ductal carcinomas, and squamous cell carcinomas.

Additionally, the compositions of the invention may be useful in thediagnosis, treatment, and/or prevention of disorders or diseases of thepenis and urethra, including inflammatory disorders, such asbalanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis,syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis,chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome,condyloma acuminatum, condyloma latum, and pearly penile papules;urethral abnormalities, such as hypospadias, epispadias, and phimosis;premalignant lesions, including Erythroplasia of Queyrat, Bowen'sdisease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, andvarrucous carcinoma; penile cancers, including squamous cell carcinomas,carcinoma in situ, verrucous carcinoma, and disseminated penilecarcinoma; urethral neoplastic disorders, including penile urethralcarcinoma, bulbomembranous urethral carcinoma, and prostatic urethralcarcinoma; and erectile disorders, such as priapism, Peyronie's disease,erectile dysfunction, and impotence.

Moreover, diseases and/or disorders of the vas deferens includevasculititis and CBAVD (congenital bilateral absence of the vasdeferens); additionally, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used in the diagnosis, treatment, and/or prevention of diseasesand/or disorders of the seminal vesicles, including hydatid disease,congenital chloride diarrhea, and polycystic kidney disease.

Other disorders and/or diseases of the male reproductive system include,for example, Klinefelter's syndrome, Young's syndrome, prematureejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome,high fever, multiple sclerosis, and gynecomastia.

Further, the polynucleotides, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused in the diagnosis, treatment, and/or prevention of diseases and/ordisorders of the vagina and vulva, including bacterial vaginosis,candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale,lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma,vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonasvaginitis, condyloma acuminatum, syphilis, molluscum contagiosum,atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus,vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvarvestibulitis, and neoplastic disorders, such as squamous cellhyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas,cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.

Disorders and/or diseases of the uterus include dysmenorrhea,retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatorybleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman'ssyndrome, premature menopause, precocious puberty, uterine polyps,dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals),and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, andsarcomas. Additionally, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful as a marker or detector of, as well as in the diagnosis,treatment, and/or prevention of congenital uterine abnormalities, suchas bicornuate uterus, septate uterus, simple unicornuate uterus,unicornuate uterus with a noncavitary rudimentary horn, unicornuateuterus with a non-communicating cavitary rudimentary horn, unicornuateuterus with a communicating cavitary horn, arcuate uterus, uterinedidelfus, and T-shaped uterus.

Ovarian diseases and/or disorders include anovulation, polycystic ovarysyndrome (Stein-Leventhal syndrome), ovarian cysts, ovarianhypofunction, ovarian insensitivity to gonadotropins, ovarianoverproduction of androgens, right ovarian vein syndrome, amenorrhea,hirutism, and ovarian cancer (including, but not limited to, primary andsecondary cancerous growth, Sertoli-Leydia tumors, endometriod carcinomaof the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinousadenocarcinoma, and Ovarian Krukenberg tumors).

Cervical diseases and/or disorders include cervicitis, chroniccervicitis, mucopurulent cervicitis, cervical dysplasia, cervicalpolyps, Nabothian cysts, cervical erosion, cervical incompetence, andcervical neoplasms (including, for example, cervical carcinoma, squamousmetaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, andcolumnar cell neoplasia).

Additionally, diseases and/or disorders of the reproductive systeminclude disorders and/or diseases of pregnancy, including miscarriageand stillbirth, such as early abortion, late abortion, spontaneousabortion, induced abortion, therapeutic abortion, threatened abortion,missed abortion, incomplete abortion, complete abortion, habitualabortion, missed abortion, and septic abortion; ectopic pregnancy,anemia, Rh incompatibility, vaginal bleeding during pregnancy,gestational diabetes, intrauterine growth retardation, polyhydramnios,HELLP syndrome, abruptio placentae, placenta previa, hyperemesis,preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy.Additionally, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused in the diagnosis, treatment, and/or prevention of diseases that cancomplicate pregnancy, including heart disease, heart failure, rheumaticheart disease, congenital heart disease, mitral valve prolapse, highblood pressure, anemia, kidney disease, infectious disease (e.g.,rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis,chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves'disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronicactive hepatitis, cirrhosis of the liver, primary biliary cirrhosis,asthma, systemic lupus eryematosis, rheumatoid arthritis, myastheniagravis, idiopathic thrombocytopenic purpura, appendicitis, ovariancysts, gallbladder disorders, and obstruction of the intestine.

Complications associated with labor and parturition include prematurerupture of the membranes, pre-term labor, post-term pregnancy,postmaturity, labor that progresses too slowly, fetal distress (e.g.,abnormal heart rate (fetal or maternal), breathing problems, andabnormal fetal position), shoulder dystocia, prolapsed umbilical cord,amniotic fluid embolism, and aberrant uterine bleeding.

Further, diseases and/or disorders of the postdelivery period, includingendometritis, myometritis, parametritis, peritonitis, pelvicthrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis,saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage,and inverted uterus.

Other disorders and/or diseases of the female reproductive system thatmay be diagnosed, treated, and/or prevented by the albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention include, for example, Turner's syndrome,pseudohermaphroditism, premenstrual syndrome, pelvic inflammatorydisease, pelvic congestion (vascular engorgement), frigidity,anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.

Infectious Disease

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention can be used to treat or detectinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated. The immune responsemay be increased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may also directly inhibit the infectious agent, withoutnecessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated or detected by albumin fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Miorbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, canbe used to treat or detect any of these symptoms or diseases. Inspecific embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g.,hepatitis B). In an additional specific embodiment fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention are used to treat patients nonresponsive to one or moreother commercially available hepatitis vaccines. In a further specificembodiment fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention are used to treatAIDS.

Similarly, bacterial and fungal agents that can cause disease orsymptoms and that can be treated or detected by albumin fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention include, but not limited to, the followingGram-Negative and Gram-positive bacteria, bacterial families, and fungi:Actinomyces (e.g., Norcardia), Acinetobacter, Cryprococcus neoformans,Aspergillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g.,Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g.,Borrelia burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia,Clostridium (e.g., Clostridium botulinum, Clostridium dificile,Clostridium perfringens, Clostridium tetani), Coccidioides,Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus,Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli andEnterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter aerogenes),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi,Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia,Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza typeB), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira,Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium(e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio(e.g. Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea,Neisseria meningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g.,Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponemaspp., Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus(e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus andStreptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and CStreptococci), and Ureaplasmas. These-bacterial, parasitic, and fungalfamilies can cause diseases or symptoms, including, but not limited to:antibiotic-resistant infections, bacteremia, endocarditis, septicemia,eye infections (e.g., conjunctivitis), uveitis, tuberculosis,gingivitis, bacterial diarrhea, opportunistic infections (e.g., AIDSrelated infections), paronychia, prosthesis-related infections, dentalcaries, Reiter's Disease, respiratory tract infections, such as WhoopingCough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery,paratyphoid fever, food poisoning, Legionella disease, chronic and acuteinflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea,meningitis (e.g., mengitis types A and B), chlamydia, syphillis,diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneousabortions, birth defects, pneumonia, lung infections, ear infections,deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea,Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatorydiseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism,gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexuallytransmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses),toxemia, urinary tract infections, wound infections, noscomialinfections. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, canbe used to treat or detect any of these symptoms or diseases. Inspecific embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to treat: tetanus, diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventioninclude, but not limited to, the following families or class: Amebiasis,Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine,Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma,Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas andSporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodiummalariae and Plasmodium ovale). These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), malaria, pregnancy complications, andtoxoplasmosis. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, canbe used to treat, prevent, and/or diagnose any of these symptoms ordiseases. In specific embodiments, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used to treat, prevent, and/or diagnose malaria.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention could either be byadministering an effective amount of an albumin fusion protein of theinvnetion to the patient, or by removing cells from the patient,supplying the cells with a polynucleotide of the present invention, andreturning the engineered cells to the patient (ex vivo therapy).Moreover, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention can beused as an antigen in a vaccine to raise an immune response againstinfectious disease.

Regeneration

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention can be used to differentiate,proliferate, and attract cells, leading to the regeneration of tissues.(See, Science 276:59-87 (1997)). The regeneration of tissues could beused to repair, replace, or protect tissue damaged by congenitaldefects, trauma (wounds, burns, incisions, or ulcers), age, disease(e.g. osteoporosis, osteocarthritis, periodontal disease, liverfailure), surgery, including cosmetic plastic surgery, fibrosis,reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may increaseregeneration of tissues difficult to heal. For example, increasedtendon/ligament regeneration would quicken recovery time after damage.Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention could also be usedprophylactically in an effort to avoid damage. Specific diseases thatcould be treated include of tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by usingfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention, to proliferate and differentiate nervecells. Diseases that could be treated using this method include centraland peripheral nervous system diseases, neuropathies, or mechanical andtraumatic disorders (e.g., spinal cord disorders, head trauma,cerebrovascular disease, and stoke). Specifically, diseases associatedwith peripheral nerve injuries, peripheral neuropathy (e.g., resultingfrom chemotherapy or other medical therapies), localized neuropathies,and central nervous system diseases (e.g., Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome), could all be treated using thealbumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention.

Gastrointestinal Disorders

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, may be used to treat, prevent,diagnose, and/or prognose gastrointestinal disorders, includinginflammatory diseases and/or conditions, infections, cancers (e.g.,intestinal neoplasms (carcinoid tumor of the small intestine,non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)),and ulcers, such as peptic ulcers.

Gastrointestinal disorders include dysphagia, odynophagia, inflammationof the esophagus, peptic esophagitis, gastric reflux, submucosalfibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas,epidermal cancers, adeoncarcinomas, gastric retention disorders,gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of thestomach, autoimmune disorders such as pernicious anemia, pyloricstenosis, gastritis (bacterial, viral, eosinophilic, stress-induced,chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritonealdiseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst,mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis,neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).

Gastrointestinal disorders also include disorders associated with thesmall intestine, such as malabsorption syndromes, distension, irritablebowel syndrome, sugar intolerance, celiac disease, duodenal ulcers,duodenitis, tropical sprue, Whipple's disease, intestinallymphangiectasia, Crohn's disease, appendicitis, obstructions of theileum, Meckel's diverticulum, multiple diverticula, failure of completerotation of the small and large intestine, lymphoma, and bacterial andparasitic diseases (such as Traveler's diarrhea, typhoid andparatyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides),Hookworms (Ancylostoma duodenale), Threadworms (Enterobiusvermicularis), Tapeworms (Taenia saginaa, Echinococcus granulosus,Diphyllobothrium spp., and T. solium).

Liver diseases and/or disorders include intrahepatic cholestasis(alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholicfatty liver, reye syndrome), hepatic vein thrombosis, hepatolentriculardegeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenalsyndrome, portal hypertension (esophageal and gastric varices), liverabscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary andexperimental), alcoholic liver diseases (fatty liver, hepatitis,cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebicliver abscess), jaundice (hemolytic, hepatocellular, and cholestatic),cholestasis, portal hypertension, liver enlargement, ascites, hepatitis(alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune,hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis,viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitisD, hepatitis E), Wilson's disease, granulomatous hepatitis, secondarybiliary cirrhosis, hepatic encephalopathy, portal hypertension, varices,hepatic encephalopathy, primary biliary cirrhosis, primary sclerosingcholangitis, hepatocellular adenoma, hemangiomas, bile stones, liverfailure (hepatic encephalopathy, acute liver failure), and liverneoplasms (angiomyolipoma, calcified liver metastases, cystic livermetastases, epithelial tumors, fibrolamellar hepatocarcinoma, focalnodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma,hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liverhemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors ofliver, nodular regenerative hyperplasia, benign liver tumors (Hepaticcysts [Simple cysts, Polycystic liver disease, Hepatobiliarycystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymalhamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis,Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors[Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma),Hepatocyte (Adenoma, Focal nodular hyperplasia. Nodular regenerativehyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma,hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma,cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi'ssarcoma, hemangioendothelioma, other tumors, embryonal sarcoma,fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosishepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittentporphyria, porphyria cutanea tarda), Zellweger syndrome).

Pancreatic diseases and/or disorders include acute pancreatitis, chronicpancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis),neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma,insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-celltumors, pancreoblastoma), and other pancreatic diseases (e.g., cysticfibrosis, cyst (pancreatic pseudocyst, pancreatic fistula,insufficiency)).

Gallbladder diseases include gallstones (cholelithiasis andcholedocholithiasis), postcholecystectomy syndrome, diverticulosis ofthe gallbladder, acute cholecystitis, chronic cholecystitis, bile ducttumors, and mucocele.

Diseases and/or disorders of the large intestine includeantibiotic-associated colitis, diverticulitis, ulcerative colitis,acquired megacolon, abscesses, fungal and bacterial infections,anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps(e.g., villous adenoma), colon carcinoma, colorectal cancer], colonicdiverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease,toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]),constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery);duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenalulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ilealdiseases (ileal neoplasms, ileitis), immunoproliferative smallintestinal disease, inflammatory bowel disease (ulcerative colitis,Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis,balantidiasis, blastocystis infections, cryptosporidiosis,dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula(rectal fistula), intestinal neoplasms (cecal neoplasms, colonicneoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps,jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferentloop syndrome, duodenal obstruction, impacted feces, intestinalpseudo-obstruction [cecal volvulus], intussusception), intestinalperforation, intestinal polyps (colonic polyps, gardner syndrome,peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms),malabsorption syndromes (blind loop syndrome, celiac disease, lactoseintolerance, short bowl syndrome, tropical sprue, whipple's disease),mesenteric vascular occlusion, pneumatosis cystoides intestinalis,protein-losing enteropathies (intestinal lymphagiectasis), rectaldiseases (anus diseases, fecal incontinence, hemorrhoids, proctitis,rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenalulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer,Zollinger-Ellison syndrome), postgastrectomy syndromes (dumpingsyndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux(bile reflux), gastric antral vascular ectasia, gastric fistula, gastricoutlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis,stomach dilatation, stomach diverticulum, stomach neoplasms (gastriccancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastricpolyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis,visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum,postoperative nausea and vomiting) and hemorrhagic colitis.

Further diseases and/or disorders of the gastrointestinal system includebiliary tract diseases, such as, gastroschisis, fistula (e.g., biliaryfistula, esophageal fistula, gastric fistula, intestinal fistula,pancreatic fistula), neoplasms (e.g., biliary tract neoplasms,esophageal neoplasms, such as adenocarcinoma of the esophagus,esophageal squamous cell carcinoma, gastrointestinal neoplasms,pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinouscystic neoplasm of the pancreas, pancreatic cystic neoplasms,pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g.,bullous diseases, candidiasis, glycogenic acanthosis, ulceration,barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker'sdiverticulum), fistula (e.g., tracheoesophageal fistula), motilitydisorders (e.g., CREST syndrome, deglutition disorders, achalasia,spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaavesyndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatichernia (e.g., hiatal hernia); Gastrointestinal diseases, such as,gastroenteritis (e.g., cholera morbus, norwalk virus infection),hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomachneoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma,stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoralhernia, inguinal hernia, obturator hernia, umbilical hernia, ventralhernia), and intestinal diseases (e.g., cecal diseases (appendicitis,cecal neoplasms)).

Chemotaxis

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may have chemotaxis activity. Achemotaxic molecule attracts or mobilizes cells (e.g., monocytes,fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelialand/or endothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may increase chemotaxicactivity of particular cells. These chemotactic molecules can then beused to treat inflammation, infection, hyperproliferative disorders, orany immune system disorder by increasing the number of cells targeted toa particular location in the body. For example, chemotaxic molecules canbe used to treat wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treatwounds.

It is also contemplated that fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayinhibit chemotactic activity. These molecules could also be used totreat disorders. Thus, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention couldbe used as an inhibitor of chemotaxis.

Binding Activity

Albumin fusion proteins of the invention may be used to screen formolecules that bind to the Therapeutic protein portion of the fusionprotein or for molecules to which the Therapeutic protein portion of thefusion protein binds. The binding of the fusion protein and the moleculemay activate (agonist), increase, inhibit (antagonist); or decreaseactivity of the fusion protein or the molecule bound. Examples of suchmolecules include antibodies, oligonucleotides, proteins (e.g.,receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of theTherapeutic protein portion of the fusion protein of the invention,e.g., a fragment of the ligand, or a natural substrate, a ligand, astructural or functional mimetic. (See, Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991)). Similarly, the moleculecan be closely related to the natural receptor to which the Therapeuticprotein portion of an albumin fusion protein of the invention binds, orat least, a fragment of the receptor capable of being bound by theTherapeutic protein portion of an albumin fusion protein of theinvention (e.g., active site). In either case, the molecule can berationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the albumin fusion proteins of theinvention. Preferred cells include cells from mammals, yeast,Drosophila, or E. coli.

The assay may simply test binding of a candidate compound to an albuminfusion protein of the invention, wherein binding is detected by a label,or in an assay involving competition with a labeled competitor. Further,the assay may test whether the candidate compound results in a signalgenerated by binding to the fusion protein.

Alternatively, the assay can be carried out using cell-freepreparations, fusion protein/molecule affixed to a solid support,chemical libraries, or natural product mixtures. The assay may alsosimply comprise the steps of mixing a candidate compound with a solutioncontaining an albumin fusion protein, measuring fusion protein/moleculeactivity or binding, and comparing the fusion protein/molecule activityor binding to a standard.

Preferably, an ELISA assay can measure fusion protein level or activityin a sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure fusion protein level or activity byeither binding, directly or indirectly, to the albumin fusion protein orby competing with the albumin fusion protein for a substrate.

Additionally, the receptor to which a Therapeutic protein portion of analbumin fusion protein of the invention binds can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting (Coligan, et al., Current Protocols in Immun.,1(2), Chapter 5, (1991)). For example, in cases wherein the Therapeuticprotein portion of the fusion protein corresponds to FGF, expressioncloning may be employed wherein polyadenylated RNA is prepared from acell responsive to the albumin fusion protein, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the albumin fusion protein. Transfected cellswhich are grown on glass slides are exposed to the albumin fusionprotein of the present invention, after they have been labeled. Thealbumin fusion proteins can be labeled by a variety of means includingiodination or inclusion of a recognition site for a site-specificprotein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, a labeledalbumin fusion protein can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule for theTherapeutoc protein component of an albumin fusion protein of theinvention, the linked material may be resolved by PAGE analysis andexposed to X-ray film. The labeled complex containing the receptors ofthe fusion protein can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino-acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the genesencoding the putative receptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of the fusionprotein, and/or Therapeutic protein portion or albumin component of analbumin fusion protein of the present invention, thereby effectivelygenerating agonists and antagonists of an albumin fusion protein of thepresent invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol.16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76(1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13(1998); each of these patents and publications are hereby incorporatedby reference). In one embodiment, alteration of polynucleotides encodingalbumin fusion proteins of the invention and thus, the albumin fusionproteins encoded thereby, may be achieved by DNA shuffling. DNAshuffling involves the assembly of tvo or more DNA segments into adesired molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides encoding albumin fusion proteins ofthe invention and thus, the albumin fusion proteins encoded thereby, maybe altered by being subjected to random mutagenesis by error-prone PCR,random nucleotide insertion or other methods prior to recombination. Inanother embodiment, one or more components, motifs, sections, parts,domains, fragments, etc., of an albumin fusion protein of the presentinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules: In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogrenetic protein (BMP)-2,BMP-4′., BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp),60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of theTherapeutic protein portion and/or albumin component of the albuminfusion proteins of the present invention. Biologically active fragmentsare those exhibiting activity similar, but not necessarily identical, toan activity of a Therapeutic protein portion and/or albumin component ofthe albumin fusion proteins of the present invention. The biologicalactivity of the fragments may include an improved desired activity, or adecreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of an albumin fusion protein ofthe present invention. An example of such an assay comprises combining amammalian fibroblast cell, an albumin fusion protein of the presentinvention, and the compound to be screened and ³[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of Fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³ [H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for the Therapeutic protien component of a fusion protine ofthe invention is incubated with a labeled fusion protein of the presentinvention in the presence of the compound. The ability of the compoundto enhance or block this interaction could then be measured.Alternatively, the response of a known second messenger system followinginteraction of a compound to be screened and the receptor is measuredand the ability of the compound to bind to the receptor and elicit asecond messenger response is measured to determine if the compound is apotential fusion protein. Such second messenger systems include but arenot limited to, cAMP guanylate cyclase, ion channels or phosphoinositidehydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting the fusionprotein/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the albumin fusion proteins of theinvention from suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compoundswhich bind to an albumin fusion protein of the invention comprising thesteps of: (a) incubating a candidate binding compound with an albuminfusion protein of the present invention; and (b) determining if bindinghas occurred. Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with an albumin fusion protein of the present invention, (b)assaying a biological activity, and (b) determining if a biologicalactivity of the fusion protein has been altered.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a component ofan albumin fusion protein of the invention.

As discussed herein, fusion proteins of the invention may be associatedwith heterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering fusion proteins of the invention (including antibodies)that are associated with heterologous polypeptides or nucleic acids. Inone example, the invention provides a method for delivering aTherapeutic protein into the targeted cell. In another example, theinvention provides a method for delivering a single stranded nucleicacid (e.g., antisense or ribozymes) or double stranded nucleic acid(e.g., DNA that can integrate into the cell's genome or replicateepisomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering an albumin fusion protein of the invention (e.g.,polypeptides of the invention or antibodies of the invention) inassociation with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the albumin fusion proteins of thepresent invention, or the polynucleotides encoding these fusionproteins, to screen for molecules which modify the activities of thealbumin fusion protein of the present invention or proteinscorresponding to the Therapeutic protein portion of the albumin fusionprotein. Such a method would include contacting the fusion protein witha selected compound(s) suspected of having antagonist or agonistactivity, and assaying the activity of the fusion protein followingbinding.

This invention is particularly useful for screening therapeuticcompounds by using the albumin fusion proteins of the present invention,or binding fragments thereof, in any of a variety of drug screeningtechniques. The albumin fusion protein employed in such a test may beaffixed to a solid support, expressed on a cell surface, free insolution, or located intracellularly. One method of drug screeningutilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the albumin fusionprotein. Drugs are screened against such transformed cells orsupernatants obtained from culturing such cells, in competitive bindingassays. One may measure, for example, the formulation of complexesbetween the agent being tested and an albumin fusion protein of thepresent invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the albumin fusionproteins of the present invention. These methods comprise contactingsuch an agent with an albumin fusion protein of the present invention ora fragment thereof and assaying for the presence of a complex betweenthe agent and the albumin fusion protein or a fragment thereof, bymethods well known in the art. In such a competitive binding assay, theagents to screen are typically labeled. Following incubation, free agentis separated from that present in bound form, and the amount of free oruncomplexed label is a measure of the ability of a particular agent tobind to the albumin fusion protein of the present invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to an albumin fusionprotein of the present invention, and is described in great detail inEuropean Patent Application 84/03564, published on Sep. 13, 1984, whichis incorporated herein by reference herein. Briefly stated, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with an albumin fusion protein of the presentinvention and washed. Bound peptides are then detected by methods wellknown in the art. Purified albumin fusion protein may be coated directlyonto plates for use in the aforementioned drug screening techniques. Inaddition, non-neutralizing, antibodies may be used to capture thepeptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding an albuminfusion protein of the present invention specifically compete with a testcompound for binding to the albumin fusion protein or fragments thereof.In this manner, the antibodies are used to detect the presence of anypeptide which shares one or more antigenic epitopes with an albuminfusion protein of the invention.

Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind albumin fusion proteins ofthe invention, and the binding molecules identified thereby. Thesebinding molecules are useful, for example, as agonists and antagonistsof the albumin fusion proteins of the invention. Such agonists andantagonists can be used, in accordance with the invention, in thetherapeutic embodiments described in detail, below.

This method comprises the steps of:

contacting an albumin fusion protein of the invention with a pluralityof molecules; and

identifying a molecule that binds the albumin fusion protein.

The step of contacting the albumin fusion protein of the invention withthe plurality of molecules may be effected in a number of ways. Forexample, one may contemplate immobilizing the albumin fusion protein ona solid support and bringing a solution of the plurality of molecules incontact with the immobilized polypeptides. Such a procedure would beakin to an affinity chromatographic process, with the affinity matrixbeing comprised of the immobilized albumin fusion protein of theinvention. The molecules having a selective affinity for the albuminfusion protein can then be purified by affinity selection. The nature ofthe solid support, process for attachment of the albumin fusion proteinto the solid support, solvent, and conditions of the affinity isolationor selection are largely conventional and well known to those ofordinary skill in the art.

Alternatively, one may also separate a plurality of polypeptides intosubstantially separate fractions comprising a subset of or individualpolypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by an albumin fusion protein of the invention, optionally inthe presence of an inducer should one be required for expression, todetermine if any selective affinity interaction takes place between thealbumin fusion protein and the individual clone. Prior to contacting thealbumin fusion protein with each fraction comprising individualpolypeptides, the polypeptides could first be transferred to a solidsupport for additional convenience. Such a solid support may simply be apiece of filter membrane, such as one made of nitrocellulose or nylon.In this manner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity for analbumin fusion protein of the invention. Furthermore, the amino acidsequence of the polypeptide having a selective affinity for an albuminfusion protein of the invention can be determined directly byconventional means or the coding sequence of the DNA encoding thepolypeptide can frequently be determined more conveniently. The primarysequence can then be deduced from the corresponding DNA sequence. If theamino acid sequence is to be determined from the polypeptide itself, onemay use microsequencing techniques. The sequencing technique may includemass spectroscopy.

In certain situations, it may be desirable to wash away any unboundpolypeptides from a mixture of an albumin fusion protein of theinvention and the plurality of polypeptides prior to attempting todetermine or to detect the presence of a selective affinity interaction.Such a wash step may be particularly desirable when the albumin fusionprotein of the invention or the plurality of polypeptides are bound to asolid support.

The plurality of molecules provided according to this method may beprovided by way of diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries which can be screened for molecules thatspecifically bind an albumin fusion protein of the invention. Manylibraries are known in the art that can be used, e.g., chemicallysynthesized libraries, recombinant (e.g., phage display libraries), andin vitro translation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., Science 251:767-773 (1991);Houghten et al., Nature 354:8/4-86 (1991); Lam et al., Nature 354:82-84(1991); Medynski, Bio/Technology 12:709-710 (1994); Gallop et al., J.Medicinal Chemistry 37(9):1233-1251 (1994); Ohlmeyer et al., Proc. Natl.Acad. Sci. USA 90:10922-10926 (1993); Erb et al., Proc. Natl. Acad. Sci.USA 91:11422-11426 (1994); Houghten et al., Biotechniques 13:412 (1992);Jayawickreme et al., Proc. Natl. Acad. Sci. USA 91:161w 1618 (1994);Salmon et al., Proc. Natl. Acad. Sci. USA 90:11708-11712 (1993); PCTPublication No. WO 93/20242; and Brenner and Lerner, Proc. Natl. Acad.Sci. USA 89:5381-5383 (1992).

Examples of phage display libraries are described in Scott et al.,Science 249:386-390 (1990); Devlin et al., Science, 249:404-406 (1990);Christian et al., 1992, J. Mol. Biol. 227:711-718 1992); Lenstra, J.Immunol. Meth. 152:149-157 (1992); Kay et al., Gene 128:59-65 (1993);and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-9026 (1994).

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., Proc. Natl. Acad. Sci. USA 91:4708-4712 (1994))can be adapted for use. Peptoid libraries (Simon et al., Proc. Natl.Acad. Sci. USA 89:9367-9371 (1992)) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (Proc. Natl. Acad.Sci. USA 91:11138-11142 (1994)).

The variety of non-peptide libraries that are useful in the presentinvention is great. For example, Ecker and Crooke (Bio/Technology13:351-360 (1995) list benzodiazepines, hydantoins, piperazinediones,biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids,acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, andoxazolones as among the chemical species that form the basis of variouslibraries.

Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers thatare assembled together in ways that create new shapes that depend on theorder of the monomers. Among the monomer units that have been used arecarbamates, pyrrolinones, and morpholinos. Peptoids, peptide-likeoligomers in which the side chain is attached to the alpha amino crouprather than the alpha carbon, form the basis of another version ofnon-peptide oligomer libraries. The first non-peptide oligomer librariesutilized a single type of monomer and thus contained a repeatingbackbone. Recent libraries have utilized more than one monomer, givingthe libraries added flexibility.

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley et al., Adv. Exp. Med.Biol. 251:215-218 (1989); Scott et al., Science 249:386-390 (1990);Fowlkes et al., BioTechniques 13:422-427 (1992); Oldenburg, et al.,Proc. Natl. Acad. Sci. USA 89:5393-5397 (1992); Yu et al., Cell76:933-945 (1994); Staudt et al., Science 241:577-580 (1.988); Bock etal., Nature 355:564-566 (1992); Tuerk et al., Proc. Natl. Acad. Sci. USA89:6988-6992 (1992); Ellington et al., Nature 355:850-852 (1992); U.S.Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No.5,198,346, all to Ladner et al.; Rebar et al., Science 263:671-673(1993); and PCT Publication No. WO 94/18318.

In a specific embodiment, screening to identify a molecule that binds analbumin fusion protein of the invention can be carried out by contactingthe library members with an albumin fusion protein of the inventionimmobilized on a solid phase and harvesting those library members thatbind to the albumin fusion protein. Examples of such screening methods,termed “panning” techniques are described by way of example in Parmleyet al., Gene 73:305-318 (1988); Fowlkes et al., BioTechniques 13:422-427(1992); PCT Publication No. WO 94/18318; and in references cited herein.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields et al., Nature 340:245-246 (1989); Chien etal., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991) can be used toidentify molecules that specifically bind to polypeptides of theinvention.

Where the binding molecule is a polypeptide, the polypeptide can beconveniently selected from any peptide library, including random peptidelibraries, combinatorial peptide libraries, or biased peptide libraries.The term “biased” is used herein to mean that the method of generatingthe library is manipulated so as to restrict one or more parameters thatgovern the diversity of the resulting collection of molecules, in thiscase peptides.

Thus, a truly random peptide library would generate a collection ofpeptides in which the probability of finding a particular amino acid ata given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage-vector with a DNA insert.

As mentioned above, in the case of a binding molecule that is apolypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, a binding polypeptide has in the range of 15-100 aminoacids, or 20-50 amino acids.

The selected binding polypeptide can be obtained by chemical synthesisor recombinant expression.

Other Activities

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention, may be employed intreatment for stimulating re-vascularization of ischemic tissues due tovarious disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. The albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may also be employed to stimulate angiogenesis and limbregeneration, as discussed above.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be employedfor treating wounds due to injuries, burns, post-operative tissuerepair, and ulcers since they are mitogenic to various cells ofdifferent origins, such as fibroblast cells and skeletal muscle cells,and therefore, facilitate the repair or replacement of damaged ordiseased tissue.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be employedstimulate neuronal growth and to treat and prevent neuronal damage whichoccurs in certain neuronal disorders or neuro-degenerative conditionssuch as Alzheimer's disease, Parkinson's disease, and AIDS-relatedcomplex. An albumin fusion protein of the invention and/orpolynucleotide encoding an albumin fusion protein of the invention mayhave the ability to stimulate chondrocyte growth, therefore, they may beemployed to enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may be also beemployed to prevent skin aging due to sunburn by stimulatingkeratinocyte growth.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be employedfor preventing hair loss, since FGF family members activate hair-formingcells and promotes melanocyte growth. Along the same lines, an albuminfusion protein of the invention and/or polynucleotide encoding analbumin fusion protein of the invention may be employed to stimulategrowth and differentiation of hematopoietic cells and bone marrow cellswhen used in combination with other cytokines.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be employedto maintain organs before transplantation or for supporting cell cultureof primary tissues. An albumin fusion protein of the invention and/orpolynucleotide encoding an albumin fusion protein of the invention mayalso be employed for inducing tissue of mesodermal origin todifferentiate in early embryos.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also increase ordecrease the differentiation or proliferation of embryonic stem cells,besides, as discussed above, hematopoietic lineage.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be used tomodulate mammalian characteristics, such as body height, weight, haircolor, eye color, skin, percentage of adipose tissue, pigmentation,size, and shape (e.g., cosmetic surgery). Similarly, an albumin fusionprotein of the invention and/or polynucleotide encoding an albuminfusion protein of the invention may be used to modulate mammalianmetabolism affecting catabolism, anabolism, processing, utilization, andstorage of energy.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may be used tochange a mammal's mental state or physical state by influencingbiorhythms, caricadic rhythms, depression (including depressivedisorders), tendency for violence, tolerance for pain, reproductivecapabilities (preferably by Activin or Inhibin-like activity), hormonalor endocrine levels, appetite, libido, memory, stress, or othercognitive qualities.

An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be used asa food additive or preservative, such as to increase or decrease storagecapabilities, fat content, lipid, protein, carbohydrate, vitamins,minerals, cofactors or other nutritional components.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the alterations detected in thepresent invention and practice the claimed methods. The following,working examples therefore, specifically point out preferred embodimentsof the present invention and are not to be construed as limiting in anyway the remainder of the disclosure.

EXAMPLES Example 1 Preparation of HA-hGH Fusion Proteins

An HA-hGH fusion protein was prepared as follows:

Cloning of hGH cDNA

The hGH cDNA was obtained from a human pituitary gland cDNA library(catalogue number HL1097v, Clontech Laboratories, Inc) by PCRamplification. Two oligonucleotides suitable for PCR amplification ofthe hGH cDNA, HGH1 and HGH2, were synthesized using an AppliedBiosystems 380B Oligonucleotide Synthesizer. (SEQ ID NO: 1) HGH1:5′-CCCAAGAATTCCCTTATCCAGGC-3′ (SEQ ID NO: 2) HGH2:5′-GGGAAGCTTAGAAGCCACAGGATCCCTCCACAG-3′

HGH 1 and HGH2 differed from the equivalent portion of the hGH cDNAsequence (Martial et. al., 1979) by two and three nucleotides,respectively, such that after PCR amplification an EcoRI site would beintroduced to the 5′ end of the cDNA and a BamHI site would beintroduced into the 3′ end of the cDNA. In addition, HGH2 contained aHindIII site immediately downstream of the hGH sequence.

PCR amplification using a Perkin-Elmer-Cetus Thermal Cycler 9600 and aPerkin-Elmer-Cetus PCR kit, was performed using single-stranded DNAtemplate isolated from the phage particles of the cDNA library asfollows: 10 μL phage particles were lysed by the addition of 10 μL phagelysis buffer (280 μg/mL proteinase K in TE buffer) and incubation at 55°C. for 15 min followed by 85° C. for 15 min. After a 1 min. incubationon ice, phage debris was pelleted by centrifugation at 14,000 rpm for 3min. The PCR mixture contained 6 μL of this DNA template, 0.1 μM of eachprimer and 200 μM of each deoxyribonucleotide. PCR was carried out for30 cycles, denaturing at 94° C. for 30 s, annealing at 65° C. for 30 sand extending at 72° C. for 30 s, increasing the extension time by 1 sper cycle.

Analysis of the reaction by gel electrophoresis showed a single productof the expected size (589 base pairs).

The PCR product was purified using Wizard PCR Preps DNA PurificationSystem (Promega Corp) and then digested with EcoRI and HindIII. Afterfurther purification of the EcoRI-HindIII fragment by gelelectrophoresis, the product was cloned into pUC19 (GIBCO BRL) digestedwith EcoRI and HindIII, to give pHGH1. DNA sequencing of the EcoRIHindIII region showed that the PCR product was identical in sequence tothe hGH sequence (Martial et al., 1979), except at the 5′ and 3′ ends,where the EcoRI and BamHI sites had been introduced, respectively.

Expression of the hGH cDNA.

The polylinker sequence of the phagemid pBluescribe (+) (Stratagrene)was replaced by inserting an oligonucleotide linker, formed by annealingtwo 75-mer oligonucleotides, between the EcoRI and HindIII sites to formpBST(+). The new polylinker included a unique NotI site.

The NotI FA expression cassette of pAYE309 (EP 431 880) comprising thePRBI promoter, DNA encoding the HA/MFα-1 hybrid leader sequence, DNAencoding HA and the ADH1 terminator, was transferred to pBST(+) to formpHA1. The HAt coding sequence was removed from this plasmid by digestionwith HindIII followed by religation to form pHA2.

Cloning of the hGH cDNA, as described in Example 1, provided the hGHcoding region lacking the pro-hGH sequence and the first 8 base pairs(bp) of the mature hGH sequence. In order to construct an expressionplasmid for secretion of hGH from yeast, a yeast promoter, signalpeptide and the first 8 bp of the hGH sequence were attached to the 5′end of the cloned hGH sequence as follows: The HindIII-SfaNI fragmentfrom pHA 1 was attached to the 5′ end of the EcoRI/HindIII fragment frompHGHI via two synthetic oligonucleotides, HGH3 and HGH4 (which cananneal to one another in such a way as to generate a double strandedfragment of DNA with sticky ends that can anneal with SfaNIand EcoRIsticky ends): (SEQ ID NO: 3) HGH3: 5′-GATAAAGATTCCCAAC-3′ (SEQ ID NO: 4)HGH4: 5′-AATTGTTGGGAATCTTT-3′

The HindIII fragment so formed was cloned into HindIII-digested pHA2 tomake pHGH2, such that the hGH cDNA was positioned downstream of the PRBIpromoter and HA/MFα-1 fusion leader sequence (see, e.g., InternationalPublication No. WO90/01063). The Notl expression cassette contained inpHGH2, which included the ADH1 terminator downstream of the hGH cDNA,was cloned into NotI-digested pSAC35 (Sleep et al., BioTechnology 8:42(1990)) to make pHGH12. This plasmid comprised the entire 2 μm plasmidto provide replication functions and the LEU2 gene for selection oftransformants.

pHGH12 was introduced into S. cerevisiae D88 by transformation andindividual transformrants were grown for 3 days at 30° C. in 10 mL YEPD(1% w/v yeast extract, 2% w/v, peptone, 2% w/v, dextrose).

After centrifugation of the cells, the supernatants were examined bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were found tocontain protein which was of the expected size and which was recognizedby anti-hGH antiserum (Sigma, Poole, UK) on Western blots.

Cloning and Expression of an HA-hGH Fusion Protein.

In order to fuse the HA cDNA to the 5′ end of the hGH cDNA, the pHA1HindIII-Bsu361 fragment (containing most of the HA cDNA) was joined tothe pHGH1 EcoRI-HindIII fragment (containing most of the hGH cDNA) viatwo oligonucleotides, HGH7 and HGH8 (SEQ ID NO: 5) HGH7:5′-TTAGGCTTATTCCCAAC 3′ (SEQ ID NO: 6) HGH8: 5′-AATTGTTGGGAATAAGCC 3′

The HindIII fragment so formed was cloned into pHA2 digested withHindIII to make pHGH10, and the NotI expression cassette of this plasmidwas cloned into NotI-digested pSAC35 to make pHGH16.

pHGH16 was used to transform S. cerevisiae D83 and supernatants ofcultures were analyzed as described above. A predominant band wasobserved that had a molecular weight of approximately 88 kD,corresponding to the combined masses of HA and hGH. Western blottingusing anti-HA and anti-hGH antisera (Sigma) confirmed the presence ofthe two constituent parts of the albumin fusion protein.

The albumin fusion protein was purified from culture supernatant bycation exchange chromatography, followed by anion exchange and gelpermeation chromatography. Analysis of the N-terminus of the protein byamino acid sequencing confirmed the presence of the expected albuminsequence.

An in vitro growth hormone activity assay (Ealey et al., GrowthRegulation 5:36 (1995)) indicated that the albumin fusion proteinpossessed full hGH activity. In a hypophysectomised rat weight gainmodel, performed essentially as described in the European Pharmacopoeia(1987, monograph 556), the fusion molecule was more potent than hGH whenthe same number of units of activity (based on the above in vitro assay)were administered daily. Further experiments in which the albumin fusionprotein was administered once every four days showed a similar overallgrowth response to a daily administration of hGH. Pharmacokineticexperiments in which ¹²⁵I-labeled protein was administered to ratsindicated an approximately ten-fold increase in circulatory half-lifefor the albumin fusion protein compared to hGH.

A similar plasmid was constructed in which DNA encoding the S.cerevisiae invertase (SUC2) leader sequence replaced the sequence forthe hybrid leader, such that the encoded leader and the junction (↓)with the HA sequence were as follows: . . . MLLQAFLFLLAGFAAKISA ↓ DAHKS. . . (SEQ ID NO:7) Invertase leader              HA sequence . . .

On introduction into S. cerevisiae DBI, this plasmid directed theexpression and secretion of the albumin fusion protein at a levelsimilar to that obtained with pHGH16. Analysis of the N-terminus of thealbumin fusion protein indicated precise and efficient cleavage of theleader sequence from the mature protein.

Cloning and Expression of an hGH-HA Fusion Protein.

In order to fuse the hGH cDNA to the 5′ end of the HA cDNA, the HA cDNAwas first altered by site-directed mutagenesis to introduce an EcoNIsite near the 5′ end of the coding region. This was done by the methodof Kunkel et al. (Methods in Enzymol. 154:367 (1987)) usingsingle-stranded DNA template prepared from pHAI and a syntheticoligonucleotide, LEU4: (SEQ ID NO: 8) LEU4: 5′-GAGATGCACACCTGAGTGAGG-3′

Site-directed mutagenesis using this oligonucleotide changed the codingsequence of the HA cDNA from Lys4 to Leu4 (K4L). However, this changewas repaired when the hGH cDNA was subsequently joined at the 5′ end bylinking the pHGH2 Notl-BamHI fragment to the EcoNI-Notl fragment of themutated pHAI, via the two oligonucleotides HGH5 and HGH6: (SEQ ID NO: 9)HGH5: 5′-GATCCTGTGGCTTCGATGCACACAAGA-3′ (SEQ ID NO: 10) HGH6:5′-CTCTTGTGTGCATCGAAGCCACAG-3′

The Notl fragment so formed was cloned into Notl-digested pSAC35 to makepHGH14. pHGH14 was used to transform S. cerevisiae D88 and supernatantsof culture were analyzed as above. A predominant band was observed thathad a molecular weight of approximately 83 kD, corresponding to thecombined masses of hGH and HA. Western blotting using anti-HA andanti-hGH antisera confirmed the presence of the two constituent parts ofthe albumin fusion protein.

The albumin fusion protein was purified from culture supernatant bycation exchange chromatography, followed by anion exchange and gelpermeation chromatography. Analysis of the N-terminus of the protein byamino acid sequencing confirmed the presence of the expected hGHsequence.

In vitro studies showed that the albumin fusion protein retained hGHactivity, but was significantly less potent than an albumin fusionprotein comprising full length HA (1-585) as the N-terminal portion andhGH as the C-terminal portion, as described above.

Construction of Plasmids for the Expression of hGH Fusions to Domains ofHA.

Fusion polypeptides were made in which the hGH molecule was fused to thefirst two domains of HA (residues 1 to 387). Fusion to the N terminus ofhGH was achieved by joining the pHA1 HindIII-Sapl fragment, whichcontained most of the coding sequence for domains 1 and 2 of HA, to thepHGHI EcoRI-HindIII fragment, via the oligonucleotides HGH 11 and HGH12: (SEQ ID NO: 11) HGH11: 5′-TGTGGAAGAGCCTCAGAATTTATTCCCAAC-3′ (SEQ IDNO: 12) HGH 12: 5′-AATTGTTGGGAATAAATTCTGAGGCTCTTCC-3′

The HindIII fragment so formed was cloned into HindIII-digested pHA2 tomake pHGH37 and the Notl expression cassette of this plasmid was clonedinto Notl-digested pSAC35.

The resulting plasmid, pHGH38, contained an expression cassette that wasfound to direct secretion of the fusion polypeptide into the supernatantwhen transformed into S. cerevisiae DB 1. Western blotting using anti-HAand anti-hGH antisera confirmed the presence of the two constituentparts of the albumin fusion protein.

The albumin fusion protein was purified from culture supernatant bycation exchange chromatography followed by gel permeationchromatography.

In vivo studies with purified protein indicated that the circulatoryhalf-life was longer than that of hGH, and similar to that of an albuminfusion protein comprising full-length HA (1-585) as the N-terminalportion and hGH as the C-terminal portion, as described above. In vitrostudies showed that the albumin fusion protein retained hGH activity.

Using a similar strategy as detailed above, an albumin fusion proteincomprising the first domain of HA (residues 1-194) as the N-terminalportion and hGH as the C-terminal portion, was cloned and expressed inS. cerevisiae DBL. Western blotting of culture supernatant using anti-HAand anti-hGH antisera confirmed the presence of the two constituentparts of the albumin fusion protein.

Fusion of HA to hGH Using a Flexible Linker Sequence

Flexible linkers, comprising repeating units of[Gly-Gly-Gly-Gly-Ser]_(n), where n was either 2 or 3, were introducedbetween the HA and hGH albumin fusion protein by cloning of theoligonucleotides HGH16, HGH17, HGH18 and HGH19: HGH16: (SEQ ID NO: 13)5′-TTAGGCTTAGGTGGCGGTGGATCCGGCGGTGGTGGATCTTTCCCAA C-3′ HGH17: (SEQ IDNO: 14) 5′-AATTGTTGGGAAAGATCCACCACCGCCGGATCCACCGCCACCTAAGC C-3′ HGH18:(SEQ ID NO: 15) 5′-TTAGGCTTAGGCGGTGGTGGATCTGGTGGCGGCGGATCTGGTGGCGGTGGATCCTTCCCAAC-3′ HGH19: (SEQ ID NO: 16)5′-AATTGTTGGGAAGGATCCACCGCCACCAGATCCGCCGCCACCAGATC CACCACCGCCTAAGCC-3′

Annealing of HGH16 with HGH17 resulted in n=2, while HGH18 annealed toHGH19 resulted in n=3. After annealing, the double-strandedoligonucleotides were cloned with the EcoRI-Bsu361 fragment isolatedfrom pHGH1 into Bsu361-digested pHGH10 to make pHGH56 (where n=2) andpHGH57 (where n=3). The Notl expression cassettes from these plasmidswere cloned into NotI-digested pSAC35 to make pHGH58 and pHGH59,respectively.

Cloning of the oligonucleotides to make pHGH56 and pHGH57 introduced aBamHI site in the linker sequences. It was therefore possible toconstruct linker sequences in which n=1 and n=4, by joining either theHindIII-BamHI fragment from pHGH56 to the BamHI-HindIII fragment frompHGH57 (making n=1), or the HindIII-BamHI fragment from pHGH57 to theBamHI-HindIII fragment from pHGH56 (making n=2). Cloning of thesefragments into the HindIII site of pHA2, resulted in pHGH60 (n=1) andpHGH61 (n=4). The Notl expression cassettes from pHGH60 and pHGH61 werecloned into Notl-digested pSAC35 to make pHGH62 and pHGH63,respectively.

Transformation of S. cerevisiae with pHGH58, pHGH59, pHGH62 and pHGH63resulted in transformants that secreted the fusion polypeptides into thesupernatant. Western blotting using anti-HA and anti-hGH antiseraconfirmed the presence of the two constituent parts of the albuminfusion proteins.

The albumin fusion proteins were purified from culture supernatant bycation exchange chromatography, followed by anion exchange and gelpermeation chromatography. Analysis of the N-termini of the proteins byamino acid sequencing confirmed the presence of the expected albuminsequence. Analysis of the purified proteins by electrospray massspectrometry confirmed an increase in mass of 315 D (n=1), 630 D (n=2),945 D (n=3) and 1260 D (n=4) compared to the HA-hGH fusion proteindescribed above. The purified protein was found to be active in vitro.

Increased Shelf-Life of HA-hGH Fusion Proteins: Methods

HA-hGH and hGH were separately diluted in cell culture media containing5% horse serum to final concentrations of 100-200 μg/ml and incubated at4, 37 or 50° C. At time zero and at weekly intervals thereafter,aliquots of the samples were tested for their biological activity in theNb2 cell proliferation assay, and the data normalized to the biologicalactivity of the control (hGH solution at time zero). In other assays hGHand HA-hGH were incubated in phosphate buffer saline in at 4, 37 and 50degree C.

Nb2 cell proliferation assay: The growth of these cells is dependent onhGH or other lactogenic hormones. In a typical experiment 10⁴ cells/wellare plated in 96-well plate in the presence of different concentrationof hGH or HA-hGH in media such as DMEM containing 5-10% horse serum for24-48 hrs in the incubator. After the incubation period, 1:10 volume ofMTT (5 mg/ml in H₂O) is added to each well and the plate is incubatedfor a further 6-16 hrs. The crowing cells convert MTT to insolubleformazan. The formazan is solublized by acidic isopropanol, and thecolor produced is measured at 570 nm on microtiter plate reader. Theextent of formazan formation reflects the level of cellularproliferation.

Increased Shelf-Life of HA-hGH Fusion Proteins: Results

The fusion of Therapeutic proteins to albumin confers stability inaqueous or other solution. FIG. 1 depicts the extended shelf-life of anHA fusion protein in terms of the biological activity of HA-hGHremaining after storage in cell culture media for up to 5 weeks at 37°C. A solution of 200 μg/ml HA-hGH was prepared in tissue culture mediacontaining 5% horse serum, and the solution incubated at 37° C. startingat time zero. At the indicated times, a sample was removed and testedfor its biological activity in the Nb2 cell assay, at 2 ng/ml finalconcentration. As shown in FIG. 1, the biological activity of HA-hGHremains essentially intact (within experimental variation) after 5 weeksof incubation at 37° C. The recombinant hGH used as control for thisexperiment lost its biological activity in the first week of theexperiment.

FIG. 2 shows the stability of K-hGH after storage in cell culture mediafor up to 3 weeks at 4, 37, or 50° C. At time zero, a solution of HA-hGHwas prepared in tissue culture media containing 5% horse serum, andincubated at 4, 37, and 50° C. At the indicated periods a sample wasremoved and assayed for its biological activity in the Nb2 cellproliferation assay, at 60 ng/ml final concentration. FL-hGH retainsover 90% of its initial activity at all temperatures tested for at least3 weeks after incubation while hGH loses its biological activity withinthe first week. This level of activity is further retained for at least7 weeks at 37° C. and 5 weeks at 50° C. These results indicate thatHA-hGH is highly stable in aqueous solution even under temperaturestress.

FIGS. 3A and 3B show the stable biological activity of HA-hGH comparedto hGH in the Nb2 cell proliferation assay. Nb2 cells were grown in thepresence of increasing concentrations of recombinant hGH or HA-hGH,added at time zero. The cells were incubated for 24 or 48 hours beforemeasuring the extent of proliferation by the MTT method. The increasedstability of HA-hGH in the assay results in essentially the sameproliferative activity at 24 hours (FIG. 3A) as at 48 hours (FIG. 3B)while hGH shows a significant reduction in its proliferative activityafter 48 hours of incubation (FIGS. 3A and 3B). Compared to hGH, theHA-hGH has lower biological potency after 1 day; the albumin fusionprotein is about 5 fold less potent than hGH. However, after 2 days theH-hGH shows essentially the same potency as hGH due to the short life ofhGH in the assay. This increase in the stability of the hGH as analbumin fusion protein has a major unexpected impact on the biologicalactivity of the protein. Although the potency of the albumin fusionproteins is slightly lower than the unfused counterparts in rapidbioassays, their biological stability results in much-higher biologicalactivity in the longer term in vitro assay or in vivo assays.

Example 2 Preparation of HA-Fusion Proteins

FIG. 4 shows a map of a plasmid (pPPC0005) that can be used as the basevector for cloning the cDNAs of therapeutic partners to form HA-fusions.For example, digestion of this vector with the restriction enzymesBsu36I/Partial HindIII will allow for the insertion of a cDNA modifiedat the 5′ end to encode the last 5 amino acids of HA including theBsu36I site and at the 3′ end to include a double stop codon and HindIIIsite. As another example, digestion of this vector with the restrictionenzymes Bsu361I, SphI allows for the insertion of a cDNA modified at the5′ end to encode the last 5 amino acids of HA including the Bsu361 siteand at the 3′ end to include a double stop codon, HindIII site and theADHI terminator sequence up to and including the SphI site.

This plasmid may easily be modified by one of skill in the art, forexample, to modify, add or delete restriction sites so that one may moreeasily clone a Therapeutic protein, or fragment or variant of into thevector for the purpose of making an albumin fusion protein of theinvention.

For example, for the purpose of making an albumin fusion protein wherethe Therapeutic moiety is placed N-terminal to the (mature) albuminprotein, restriction sites were added at the 5′ end of the DNA encodingHA in pPPC0005, shown in FIG. 4).

Because it was desired to add unique XhoI and ClaI sites at the 5′ endof the DNA encoding the HA protein in pPPC0005, it was first necessaryto remove those same sites from the plasmid (located 3′ of the ADH1terminator sequence). This was accomplished by cutting pPPC0005 withXhoI and ClaI, filling in the sticky ends with T4 DNA polymerase, andreligating the blunt ends to create pPPC006 Engineering the XhoI andClaI restriction sites into the Fusion leader sequence just 5′ of theDNA encoding the HA protein in pPPC0006 was accomplished using tworounds of PCR. The first pair of oligonucleotides are those of SEQ IDNO:19 and SEQ ID NO:20. SEQ ID 19 contains four point mutations relativeto the DNA sequence encoding the Fusion leaadr sequence and thebeginning of the HA protein. These mutations are necessary to create theXhoI site in the fusion leader sequence and the Cla I site just at thebeginning of the DNA encoding the HA protein. These four mutations areunderlined in the sequence shown below. In pPPC0006 the nucleotides atthese four positions from 5′ to 3′ are T, G, T, and G. (SEQ ID NO: 19)5′-GCCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCGATTT AAAGATTTGGG-3′ (SEQID NO: 20) 5′-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAATAAGC-3′.A second round of PCR is then performed with an upstream flankingprimer, 5′-TACAAACTTAAGAGTCCAATTAGC-3′ (SEQ ID NO:21) and a downstreamflanking primer 5′-CACTTCTCTAGAGTGGTTTCATATGTCTT-3′ (SEQ ID NO:22). Theresulting PCR product is then purified and then divested with AflI andXbaI and ligated into the same sites in pPPC0006 creating pScCHSA. Theresulting plasmid will have an XhoI sites engineered into the fusionleader sequence. The presence of the XhoI site creates a single aminoacid change in the end of fusion leader sequence from LDKR to LEKR. TheD to E change will not be present in the final albumin fusion proteinexpression plasmid if one ligates into the XhoI and Cla I sites afragment comprising the Therapeutic moiety which has a 5′ SalI stickyend (which is compatible with the XhoI end) and a 3′ ClaI end. Ligationof the XhoI to the Sail restores the original amino acid sequence of theFusion leader sequence. The Therapeutic protein moiety may be insertedafter the Kex2 site (Kex2 claeves after the dibasic amino acid sequenceKR at the end of the Fusion leader sequence) and before the ClaI site.

In addition, for the purpose of making an albumin fusion protein wherethe Therapeutic moiety is placed C-terminal to the (mature) albuminprotein, four, eight-base-pair restriction sites were added at the 3′end of the DNA encoding HA in pScCHSA. As an example, it was felt to bedesirable to incorporate AscI, FseI, and PmeI restriction sites inbetween the Bsu36I and HindIII sites at the end of the DNA encoding theHA protein in pScCHSA. This was accomplished through the use of twocomplementary synthetic oligonucleotides (SEQ ID NO:19 and SEQ ID NO:20)which contain the desired restriction sites. (SEQ ID NO: 23)5′-AAGCTGCCTTAGGCTTATAATAAGGCGCGCCGGCCGGCCGTTTAAAC TAAGCTTAATTCT-3′ and(SEQ ID NO: 24) 5′-AGAATTAAGCTTAGTTTAAACGGCCGGCCGGCGCGCCTTATTATAAGCCTAAGGCAGCTT-3′.These oligonucleotides may be annealed and digested with Bsu36I andHindIII and ligated into the same sites located at the end of the DNAencoding the HA protein in pScCHSA creating pScNHSA, using techniquesknown in the art.Making Vectors Comprising Albumin Fusion Proteins where the AlbuminMoiety is N-terminal to the Therapeutic Moiety.

The DNA encoding the Therapeutic moiety may be PCR amplified usingprimers that will add DNA encoding the last five amino acids of the HA(and containing the Bsu36I site) onto the 5′ end of the DNA encoding aTherapeutic protein and a STOP codon and appropriate cloning sites ontothe 3′ end of the coding sequence. For instance, the forward primer usedto amplify the DNA encoding a Therapeutic protein might have thesequence, 5′-aagctGCCTTAGGCTTA(N)₁₅-3′ (SEQ ID NO:25) where theunderlined sequence is a Bsu361 site, the upper case nucleotides encodethe last four amino acids of the mature HA protein (ALGL), and (N)₁₅ isidentical to the first 15 nucleotides encoding the Therapetic protein ofinterest. Similarly, the reverse primer used to amplify the DNA encodinga Therapeutic protein might have the sequence,

where the italicized nucleotides is a PmeI site, the double underlinednucleotides are a FseI site, the singly underlined text is a PmeI site,the boxed nucleotides are the reverse complement of two tandem stopcodons, and (N)₁₅ is identical to the reverse complement of the last 15nucleotides encoding the Therapeutic protein of interest. Once the PCRproduct is amplified it may be cut with Bsu36I and one of (AscI, FseI,or PmeI) and ligated into pScNHSA.Making Vectors Comprising Albumin Fusion Proteins where the AlbuminMoiety is N-Terminal to the Therapeutic Moiety.

The DNA encoding the Therapeutic moiety may be PCR amplified usingprimers that will add DNA encoding the last three amino acids of theFusion leader sequence (and containing a SalI site) onto the 5′ end ofthe DNA encoding a Therapeutic protein and the first few amino acids ofthe HLA (and containing a ClaI site. For instance, the forward primerused to amplify the DNA encoding a Therapeutic protein might have thesequence, 5′-aggagcgtcGACAAAAGA(N)₁₅-3′ (SEQ ID NO:27) where theunderlined sequence is a Sal I site, the upper case nucleotides encodethe last three amino acids of the Fusion leader sequence (DKR), and(N)₁₅ is identical to the first 15 nucleotides encoding the Therapeticprotein of interest. Similarly, the reverse primer used to amplify theDNA encoding a Therapeutic protein might have the sequence, (SEQ ID NO:28) 5′-CTTTAAATCG AT GAGCAACCTCACTCTTGTGTGCATC(N)₁₅-3′where the italicized nucleotides are a ClaI site, the underlinednucleotides are the reverse complement of the DNA encoding the first 9amino acids of HA (DAHKSEVAH), and (N) is identical to the reversecomplement of the last 15 nucleotides encoding the Therapeutic proteinof interest. Once the PCR product is amplified it may be cut with SalIand ClaI and ligated into pScCHSA digested with XhoI and Cla I.Expression of an Albumin Fusion Protein in Yeast.

The Not I fragment containing the DNA encoding either an N-terminal orC-terminal albumin fusion protein generated from pScCHSA or pScNHSA maythen be cloned in to the NotI site of pSAC35.

Expression of an Albumin Fusion Protein from Mammalian Cell Lines

The HSA gene has also been cloned into a the pC4 vector which is moresuitable for mammalian culture systems creating plasmid pC4:HSA. Morespecifically, pC4HSA was generated by PCR amplifying the mature HSA genewith a 5′ primer (SEQ ID NO:30) that anneals to the 5′ end of DNAencoding the mature form of the HSA protein (e.g, DNA in plasmidpScCHSA), incorporates BamHI (Shown in italics below) and HindIII (shownsingly underlined below) cloning sites, attaches a kozak sequence (showndouble underlined below) and DNA encoding the natural HSA signal peptide(MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:29) (shown in bold below), and a 3′primer (SEQ ID NO: 31) that anneals to the 3′ end of DNA encoding themature form of the HSA protein and incorporates an Asp711 restrictionsite (shown in bold below). The DNA encoding the natural human serumalbumin leader sequence in SEQ ID NO:30 also contains a modificationthat introduces a XhoI site that is boxed below.

This PCR product (1.85 kb) is then purified and digested with BamHI andAsp718 and cloned into the same sites in pC4 (ATCC Accession No. 209646)to produce pC4:HSAMaking Vectors Comprising Albumin Fusion Proteins where the AlbuminMoiety is C-Terminal to the Therapeutic Moiety Using the pC4:HSA Vector

Using pC4:HSA, albumin fusion proteins in which the Therapeutic proteinmoiety is N terminal to the albumin sequence, one can clone DNA encodinga Therapeutic protein that has its own signal sequence between the BamHI (or HindIII) and ClaI sites. When cloning into either the BamHI orHind III site remember to include Kozak sequence (CCGCCACCATG) prior totranslational start codon of DNA encoding the Therapeutic Protein to besubcloned. If the Therapeutic does not have a signal sequence, the DNAencoding that Therapeutic protein may be cloned in between the XhoI andClaI sites. When using the XhoI site, the following 5′ (SEQ. ID NO:32)and 3′ (SEQ ID NO:33) PCR primers may be used: (SEQ ID NO: 32)5′-CCGCCGCTCGAGGGGTGTGTTTCGTCGA(N)₁₈-3′ (SEQ ID NO: 33)5′-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATC(N)₁₈-3′

In SEQ ID NO: 32, the underlined sequence is an XhoI site; and the XhoIsite and the DNA following the XhoI site encode for the last seven aminoacids of the leader sequence of natural human serum albumin. In SEQ IDNO:33, the underlined sequence is a ClaI site; and

the ClaI site and the DNA following it encode are the reverse complementof the DNA encoding the first 10 amino acids of the mature HSA protein(SEQ ID NO: 18). In SEQ ID NO:32 “(N)₁₈” is DNA identical to the firstIS nucleotides encoding the Therapeutic protein of interest.). In SEQ IDNO:33 “(N)₁₈” is the reverse complement of DNA encoding the last 18nucleotides encoding the Therapeutic protein of interest. Using, thesetwo primers, one may PCR amplify the Therapeutic protein of interest,purify the PCR product, digest it with XhoI and ClaI restriction enzymesand then and clone it into the with XhoI and ClaI sites in the pC4:HSAvector.

Making Vectors Comprising Albumin Fusion Proteins where the AlbuminMoiety is N-Terminal to the Therapeutic Moiety Using the pC4:HSA Vector

Using pC4:HSA, albumin fusion proteins in which the Therapeutic proteinmoiety is N terminal to the albumin sequence, one can clone DNA encodinga Therapeutic protein between the Bsu36I and AscI restriction sites.When cloning into the Bsu36I and AscI, the same primer design used toclone in the yeast vector system (SEQ ID NO:25 and 26) may be employed.

The pC4 vector is especially suitable for expression of albumin fusionproteins from CHO cells. For expression, in other mammalian cell types,e.g., NSO cells, it may be useful to subclone the HindIII-EcoRI fragmentcontaining the DNA encoding an albumin fusion protein (from a pC4 vectorin which the DNA encoding the Therapeutic protein has already beencloned in frame with the DNA encoding (the mature form of) human serumalbumin) into another expression vector (such as any of the mammalianexpression vectors described herein).

Example 3 Preparation of HA-Cytokine or HA-Growth Factor Fusion Proteins(such as EPO, GMCSF, GCSF)

The cDNA for the cytokine or growth factor of interest, such as EPO, canbe isolated by a variety of means including from cDNA libraries, byRT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. The nucleotidesequences for all of these proteins are known and available, forinstance, in U.S. Pat. Nos. 4,703,008, 4,810,643 and 5,908,763. The cDNAcan be tailored at the 5′ and 3′ ends to generate restriction sites,such that oligonucleotide linkers can be used, for cloning of the cDNAinto a vector containing the cDNA for HA. This can be at the N orC-terminus with or without the use of a spacer sequence. EPO (or othercytokine) cDNA is cloned into a vector such as pPPC0005 (FIG. 4),pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassetteis then excised and inserted into the plasmid pSAC35 to allow theexpression of the albumin fusion protein in yeast. The albumin fusionprotein secreted from the yeast can then be collected and purified fromthe media and tested for its biological activity. For expression inmammalian cell lines, a similar procedure is adopted except that theexpression cassette used employs a mammalian promoter, leader sequenceand terminator (See Example 2). This expression cassette is then excisedand inserted into a plasmid suitable for the transfection of mammaliancell lines.

Example 4 Preparation of HA-IFN fusion proteins (such as IFNα)

The cDNA for the interferon of interest such as IFNα can be isolated bya variety of means including but not exclusively, from cDNA libraries,by RT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. The nucleotidesequences for interferons, such as IFNα are known and available forinstance, in U.S. Pat. Nos. 5,326,859 and 4,588,585, in EP 32 134, aswell as in public databases such as GenBank. The cDNA can be tailored atthe 5′ and 3′ ends to generate restriction sites, such thatoligonucleotide linkers can be used to clone the cDNA into a vectorcontaining the cDNA for HA. This can be at the N or C-terminus of the RNsequence, with or without the use of a spacer sequence. The IFNα (orother interferon) cDNA is cloned into a vector such as pPPC0005 (FIG.4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expressioncassette is then excised and inserted into the plasmid pSAC35 to allowthe expression of the albumin fusion protein in yeast (see FIG. 8). Thealbumin fusion protein secreted from the yeast can then be collected andpurified from the media and tested for its biological activity. Forexpression in mammalian cell lines a similar procedure is adopted exceptthat the expression cassette used employs a mammalian promoter, leadersequence and terminator (See Example 2). This expression cassette isthen excised and inserted into a plasmid suitable for the transfectionof mammalian cell lines.

Maximum Protein Recovery from Vials

The albumin fusion proteins of the invention have a high degree ofstability even when they are packaged at low concentrations. Inaddition, in spite of the low protein concentration, good fusion-proteinrecovery is observed even when the aqueous solution includes no otherprotein added to minimize binding to the vial walls. FIG. 5 compares therecovery of vial-stored FL-IFN solutions with a stock solution. 6 or 30μg/ml HA-IFN solutions were placed in vials and stored at 4° C. After 48or 72 hrs a volume originally equivalent to 10 ng of sample was removedand measured in an IFN sandwich ELISA. The estimated values werecompared to that of a high concentration stock solution. As shown, thereis essentially no loss of the sample in these vials, indicating thataddition of exogenous material such as albumin is not necessary toprevent sample loss to the wall of the vials

In Vivo Stability and Bioavailability of HA-α-IFN Fusions

To determine the in vivo stability and bioavailability of a HA-α-IFNfusion molecule, the purified fusion molecule (from yeast) wasadministered to monkeys at the dosages and time points described inFIGS. 6 and 7. Pharmaceutical compositions formulated from HA-α-IFNfusions may account for the extended serum half-life and bioavailabilityexemplified in FIGS. 6 and 7. Accordingly, pharmaceutical compositionsmay be formulated to contain lower dosages of alpha-interferon activitycompared to the native alpha-interferon molecule.

Pharmaceutical compositions containing HA-α-IFN fusions may be used totreat or prevent disease in patients with any disease or disease statethat can be modulated by the administration of α-IFN. Such diseasesinclude, but are not limited to, hairy cell leukemia, Kaposi's sarcoma,genital and anal warts, chronic hepatitis B, chronic non-A, non-Bhepatitis, in particular hepatitis C, hepatitis D, chronic myelogenousleukemia, renal cell carcinoma, bladder carcinoma, ovarian and cervicalcarcinoma, skin cancers, recurrent respirator papillomatosis,non-Hodgkin's and cutaneous T-cell lymphomas, melanoma, multiplemyeloma, AIDS, multiple sclerosis, gliobastoma, etc. (see InterferonAlpha, In: AHFS Drug Information, 1997.

Accordingly, the invention includes pharmaceutical compositionscontaining a HA-α-IFN fusion protein, polypeptide or peptide formulatedwith the proper dosage for human administration. The invention alsoincludes methods of treating patients in need of such treatmentcomprising at least the step of administering a pharmaceuticalcomposition containing at least one H-α-IFN fusion protein, polypeptideor peptide.

Bifunctional HA-_-IFN Fusions

The HA-α-IFN expression vector of FIG. 8 is modified to include aninsertion for the expression of bifunctional HA-α-IFN fusion proteins.For instance, the cDNA for a second protein of interest may be insertedin frame downstream of the “rHA-IFN” sequence after the double stopcodon has been removed or shifted downstream of the coding sequence.

In one version of a bifunctional HA-α-IFN fusion protein, an antibody orfragment against B-lymphocyte stimulator protein (GenBank Acc 4455139)or polypeptide may be fused to one end of the HA component of the fusionmolecule. This bifunctional protein is useful for modulating any immuneresponse generated by the α-IFN component of the fusion.

Example 5 Preparation of HA-Hormone Fusion Protein (Such as Insulin, LH,FSH)

The cDNA for the hormone of interest such as insulin can be isolated bya variety of means including but not exclusively, from cDNA libraries,by RT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. The nucleotidesequences for all of these proteins are known and available, forinstance, in public databases such as GenBank. The cDNA can be tailoredat the 5′ and 3′ ends to generate restriction sites, such thatoligonucleotide linkers can be used, for cloning of the cDNA into avector containing the cDNA for HA. This can be at the N or C-terminuswith or without the use of a spacer sequence. The hormone cDNA is clonedinto a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSAfrom which the complete expression cassette is then excised and insertedinto the plasmid pSAC35 to allow the expression of the albumin fusionprotein in yeast. The albumin fusion protein secreted from the yeast canthen be collected and purified from the media and tested for itsbiological activity. For expression in mammalian cell lines a similarprocedure is adopted except that the expression cassette used employs amammalian promoter, leader sequence and terminator (See Example 2). Thisexpression cassette is then excised and inserted into a plasmid suitablefor the transfection of mammalian cell lines.

Example 6 Preparation of HA-Soluble Receptor or HA-Binding ProteinFusion Protein Such as HA-TNF Receptor

The cDNA for the soluble receptor or binding protein of interest such asTNF receptor can be isolated by a variety of means including but notexclusively, from cDNA libraries, by RT-PCR and by PCR using a series ofoverlapping synthetic oligonucleotide primers, all using standardmethods. The nucleotide sequences for all of these proteins are knownand available, for instance, in GenBank. The cDNA can be tailored at the5′ and 3′ ends to generate restriction sites, such that oligonucleotidelinkers can be used, for cloning of the cDNA into a vector containingthe cDNA for HA. This can be at the N or C-terminus with or without theuse of a spacer sequence. The receptor cDNA is cloned into a vector suchas pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which thecomplete expression cassette is then excised and inserted into theplasmid pSAC35 to allow the expression of the albumin fusion protein inyeast. The albumin fusion protein secreted from the yeast can then becollected and purified from the media and tested for its biologicalactivity. For expression in mammalian cell lines a similar procedure isadopted except that the expression cassette used employs a mammalianpromoter, leader sequence and terminator (See Example 2). Thisexpression cassette is then excised and inserted into a plasmid suitablefor the transfection of mammalian cell lines.

Example 7 Preparation of HA-Growth Factors Such as HA-IGF-1 FusionProtein

The cDNA for the growth factor of interest such as IGF-1 can be isolatedby a variety of means including but not exclusively, from cDNAlibraries, by RT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods (see GenBank Acc.No. NP_(—)000609). The cDNA can be tailored at the 5′ and 3′ ends togenerate restriction sites, such that oligonucleotide linkers can beused, for cloning of the cDNA into a vector containing the cDNA for HA.This can be at the N or C-terminus with or without the use of a spacersequence. The growth factor cDNA is cloned into a vector such aspPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the completeexpression cassette is then excised and inserted into the plasmid pSAC35to allow the expression of the albumin fusion protein in yeast. Thealbumin fusion protein secreted from the yeast can then be collected andpurified from the media and tested for its biological activity. Forexpression in mammalian cell lines a similar procedure is adopted exceptthat the expression cassette used employs a mammalian promoter, leadersequence and terminator (See Example 2). This expression cassette isthen excised and inserted into a plasmid suitable for the transfectionof mammalian cell lines.

Example 8 Preparation of HA-Single Chain Antibody Fusion Proteins

Single chain antibodies are produced by several methods including butnot limited to: selection from phage libraries, cloning of the variableregion of a specific antibody by cloning the cDNA of the antibody andusing the flanking constant regions as the primer to clone the variableregion, or by synthesizing an oligonucleotide corresponding to thevariable region of any specific antibody. The cDNA can be tailored atthe 5′ and 3′ ends to generate restriction sites, such thatoligonucleotide linkers can be used, for cloning of the cDNA into avector containing the cDNA for HA. This can be at the N or C-terminuswith or without the use of a spacer sequence. The cell cDNA is clonedinto a vector such as pPPC005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSAfrom which the complete expression cassette is then excised and insertedinto the plasmid pSAC35 to allow the expression of the albumin fusionprotein in yeast.

In fusion molecules of the invention, the V_(H) and V_(L) can be linkedby one of the following means or a combination thereof: a peptide linkerbetween the C-terminus of the V_(H) and the N-terminus of the V_(L); aKex2p protease cleavage site between the V_(H) and V_(L) such that thetwo are cleaved apart upon secretion and then self associate; andcystine residues positioned such that the V_(H) and V_(L) can form adisulphide bond between them to link them together (see FIG. 14). Analternative option would be to place the V at the N-terminus of HA or anHA domain fragment and the V_(L) at the C-terminus of the HA or HAdomain fragment.

The albumin fusion protein secreted from the yeast can then be collectedand purified from the media and tested for its activity. For expressionin mammalian cell lines a similar procedure is adopted except that theexpression cassette used employs a mammalian promoter, leader sequenceand terminator (See Example 2). This expression cassette is then excisedand inserted into a plasmid suitable for the transfection of mammaliancell lines. The antibody produced in this manner can be purified frommedia and tested for its binding to its antigen using standardimmunochemical methods.

Example 9 Preparation of HA-Cell Adhesion Molecule Fusion Proteins

The cDNA for the cell adhesion molecule of interest can be isolated by avariety of means including but not exclusively, from cDNA libraries, byRT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. The nucleotidesequences for the known cell adhesion molecules are known and availablefor instance, in GenBank. The cDNA can be tailored at the 5′ and 3′ endsto generate restriction sites, such that oligonucleotide linkers can beused, for cloning of the cDNA into a vector containing the cDNA for FLA.This can be at the N or C-terminus with or without the use of a spacersequence. The cell adhesion molecule cDNA is cloned into a vector suchas pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which thecomplete expression cassette is then excised and inserted into theplasmid pSAC35 to allow the expression of the albumin fusion protein inyeast. The albumin fusion protein secreted from the yeast can then becollected and purified from the media and tested for its biologicalactivity. For expression in mammalian cell lines a similar procedure isadopted except that the expression cassette used employs a mammalianpromoter, leader sequence and terminator (See Example 2). Thisexpression cassette is then excised and inserted into a plasmid suitablefor the transfection of mammalian cell lines.

Example 10 Preparation of Inhibitory Factors and Peptides as HA FusionProteins (Such as HA-Antiviral, HA-Antibiotic, HA-Enzyme Inhibitor andHA-Anti-Allergic Proteins)

The cDNA for the peptide of interest such as an antibiotic peptide canbe isolated by a variety of means including but not exclusively, fromcDNA libraries, by RT-PCR and by PCR using a series of overlappingsynthetic oligonucleotide primers, all using standard methods. The cDNAcan be tailored at the 5′ and 3′ ends to generate restriction sites,such that oligonucleotide linkers can be used, for cloning of the cDNAinto a vector containing the cDNA for HA. This can be at the N orC-terminus with or without the use of a spacer sequence. The peptidecDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA.pScNHSA, or pC4:HSA from which the complete expression cassette is thenexcised and inserted into the plasmid pSAC35 to allow the expression ofthe albumin fusion protein in yeast. The albumin fusion protein secretedfrom the yeast can then be collected and purified from the media andtested for its biological activity. For expression in mammalian celllines a similar procedure is adopted except that the expression cassetteused employs a mammalian promoter, leader sequence and terminator (SeeExample 2). This expression cassette is then excised and inserted into aplasmid suitable for the transfection of mammalian cell lines.

Example 11 Preparation of Targeted HA Fusion Proteins

The cDNA for the protein of interest can be isolated from cDNA libraryor can be made synthetically using several overlapping oligonucleotidesusing standard molecular biology methods. The appropriate nucleotidescan be engineered in the cDNA to form convenient restriction sites andalso allow the attachment of the protein cDNA to albumin cDNA similar tothe method described for hGH. Also a targeting protein or peptide cDNAsuch as single chain antibody or peptides, such as nuclear localizationsignals, that can direct proteins inside the cells can be fused to theother end of albumin. The protein of interest and the targeting peptideis cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, orpC4:HSA which allows the fusion with albumin cDNA. In this manner bothN- and C-terminal end of albumin are fused to other proteins. The fusedcDNA is then excised from pPPC0005 and is inserted into a plasmid suchas pSAC35 to allow the expression of the albumin fusion protein inyeast. All the above procedures can be performed using standard methodsin molecular biology. The albumin fusion protein secreted from yeast canbe collected and purified from the media and tested for its biologicalactivity and its targeting activity using appropriate biochemical andbiological tests.

Example 12 Preparation of HA-Enzymes Fusions

The cDNA for the enzyme of interest can be isolated by a variety ofmeans including but not exclusively, from cDNA libraries, by RT-PCR andby PCR using a series of overlapping synthetic oligonucleotide primers,all using standard methods. The cDNA can be tailored at the 5′ and 3′ends to generate restriction sites, such that oligonucleotide linkerscan be used, for cloning of the cDNA into a vector containing the cDNAfor H.L. This can be at the N or C-terminus with or without the use of aspacer sequence. The enzyme cDNA is cloned into a vector such aspPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the completeexpression cassette is then excised and inserted into the plasmid pSAC35to allow the expression of the albumin fusion protein in yeast. Thealbumin fusion protein secreted from the yeast can then be collected andpurified from the media and tested for its biological activity. Forexpression in mammalian cell lines a similar procedure is adopted exceptthat the expression cassette used employs a mammalian promoter, leadersequence and terminator (See Example 2). This expression cassette isthen excised and inserted into a plasmid suitable for the transfectionof mammalian cell lines.

Example 13 Bacterial Expression of an Albumin Fusion Protein

A polynucleotide encoding an albumin fusion protein of the presentinvention comprising a bacterial signal sequence is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ ends of the DNAsequence, to synthesize insertion fragments. The primers used to amplifythe polynucleotide encoding insert should preferably contain restrictionsites, such as BamHI and XbaI, at the 5′ end of the primers in order toclone the amplified product into the expression vector. For example,BamHI and XbaI correspond to the restriction enzyme sites on thebacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.).This plasmid vector encodes antibiotic resistance (Amp^(r)), a bacterialorigin of replication (ori), an IPTG-regulatable promoter/operator(P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), andrestriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, which expresses the lacI repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are Crown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The ON culture is used to inoculate a large culture at aratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lad repressor, clearing the P/O leading to increasedgene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl or preferably in 8 M urea andconcentrations greater than 0.14 M 2-mercaptoethanol by stirring for 3-4hours at 4° C. (see, e.g., Burton et al., Eur. J. Biochem. 179:379-387(1989)). The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8. The column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. Exemplary conditions are asfollows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20%glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

In addition to the above expression vector, the present inventionfurther includes an expression vector, called pHE4a (ATCC AccessionNumber 209645, deposited on Feb. 25, 1998) which contains phage operatorand promoter elements operatively linked to a polynucleotide encoding analbumin fusion protein of the present invention, called pHE4a. (ATCCAccession Number 209645, deposited on Feb. 25, 1998.) This vectorcontains: 1) a neomycinphosphotransferase gene as a selection marker, 2)an E. coli origin of replication, 3) a T5 phage promoter sequence, 4)two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) thelactose operon repressor gene (lacIq). The origin of replication (oriC)is derived from pUC19 (LTI, Gaithersburg, MID). The promoter andoperator sequences are made synthetically.

DNA can be inserted into the pHE4a by restricting the vector with NdeIand XbaI, BamHI, XhoI, or Asp718, running the restricted product on agel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to PCRprotocols described herein or otherwise known in the art, using PCRprimers having restriction sites for NdeI (5′ primer) and XbaI, BamHI,XhoI, or Asp718 (3′ primer). The PCR insert is gel purified andrestricted with compatible enzymes. The insert and vector are ligatedaccording to standard protocols.

The engineered vector may be substituted in the above protocol toexpress protein in a bacterial system.

Example 14 Expression of an Albumin Fusion Protein in Mammalian Cells

The albumin fusion proteins of the present invention can be expressed ina mammalian cell. A typical mammalian expression vector contains apromoter element, which mediates the initiation of transcription ofmRNA, a protein coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription is achieved with the early and late promotersfrom SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as, pSVL and pMSG (Pharmacia,Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI(ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cellsthat could be used include, but are not limited to, human Hela, 293, H9and Jurkat cells, mouse NIH3T3 and CV127 cells, Cos 1, Cos 7 and CV1,quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the albumin fusion protein can be expressed in stablecell lines containing the polynucleotide encoding the albumin fusionprotein integrated into a chromosome. The co-transfection with aselectable marker such as DHFR, gpt, neomycin, or hygromycin allows theidentification and isolation of the transfected cells.

The transfected polynucleotide encoding the fusion protein can also beamplified to express large amounts of the encoded fusion protein. TheDHFR (dihydrofolate reductase) marker is useful in developing cell linesthat carry several hundred or even several thousand copies of the geneof interest. (See, e.g., Alt et al., J. Biol. Chem. 253: 1357-1370(1978); Hamlin et al., Biochem. et Biophys. Acta, 1097:107-143 (1990);Page et al., Biotechnology 9:64-68 (1991)). Another useful selectionmarker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992).Using these markers, the mammalian cells are grown in selective mediumand the cells with the highest resistance are selected. These cell linescontain the amplified gene(s) integrated into a chromosome. Chinesehamster ovary (CHO) and NSO cells are often used for the production ofproteins.

Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), theexpression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No. 209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors also contain the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene,and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6, for example, is digested with appropriaterestriction enzymes and then dephosphorylated using calf intestinalphosphates by procedures known in the art. The vector is then isolatedfrom a 1% agarose gel.

A polynucleotide encoding an albumin fusion protein of the presentinvention is generated using techniques known in the art and thispolynucleotide is amplified using PCR technology known in the art. If anaturally occurring signal sequence is used to produce the fusionprotein of the present invention, the vector does not need a secondsignal peptide. Alternatively, if a naturally occurring signal sequenceis not used, the vector can be modified to include a heterologous signalsequence. (See, e.g., International Publication No. WO 96/34891.)

The amplified fragment encoding the fusion protein of the invention isisolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then isdigested with appropriate restriction enzymes and again purified on a 1%agarose gel.

The amplified fragment encoding the albumin fusion protein of theinvention is then digested with the same restriction enzyme and purifiedon a 1% agarose gel. The isolated fragment and the dephosphorylatedvector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Bluecells are then transformed and bacteria are identified that contain thefragment inserted into plasmid pC6 using, for instance, restrictionenzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransfection. Five μg of the expression plasmid pC6 or pC4 iscotransfected with 0.5 μg of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 μM. Expression of the desired fusion protein isanalyzed, for instance, by SDS-PAGE and Western blot or by reversedphase HPLC analysis.

Example 15 Multifusion Fusions

The albumin fusion proteins (e.g., containing a Therapeutic protein (orfragment or variant thereof) fused to albumin (or a fragment or variantthereof)) may additionally be fused to other proteins to generate“multifusion proteins”. These multifusion proteins can be used for avariety of applications. For example, fusion of the albumin fusionproteins of the invention to His-tag, HA-tag, protein A, IgG domains,and maltose binding protein facilitates purification. (See e.g,. EP A394,827; Traunecker et al., Nature 331:8486 (1988)). Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof an albumin fusion protein. Furthermore, the fusion of additionalprotein sequences to the albumin fusion proteins of the invention mayfurther increase the solubility and/or stability of the fusion protein.The fusion proteins described above can be made using or routinelymodifting techniques known in the art and/or by modifying the followingprotocol, which outlines the fusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian or yeast expression vector.

For example, if pC4 (ATCC Accession No. 209646) is used, the human Fcportion can be ligated into the BamHI cloning site. Note that the 3′BamHI site should be destroyed. Next, the vector containing the human Fcportion is re-restricted with BamHI, linearizing the vector, and apolynucleotide encoding an albumin fusion protein of the presentinvention (generateed and isolated using techniques known in the art),is ligated into this BamHI site. Note that the polynucleotide encodingthe fusion protein of the invention is cloned without a stop codon,otherwise a Fc containing fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce thealbumin fusion protein of the present invention, pC4 does not need asecond signal peptide. Alternatively, if the naturally occurring signalsequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., International Publication No.WO 96/34891.) Human IgG Fc region: (SEQ ID NO: 36)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 16 Production of an Antibody from an Albumin Fusion Protein

a) Hybridoma Technology

Antibodies that bind the albumin fusion proteins of the presentinvention and portions of the albumin fusion proteins of the presentinvention (e.g., the Therapeutic protein portion or albumin portion ofthe fusion protein) can be prepared by a variety of methods. (See,Current Protocols, Chapter 2.) As one example of such methods, apreparation of an albumin fusion protein of the invention or a portionof an albumin fusion protein of the invention is prepared and purifiedto render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

Monoclonal antibodies specific for an albumin fusion protein of theinvention, or a portion of an albumin fusion protein of the invention,are prepared using hybridoma technology (Kohler et al., Nature 256:495(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.,Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).In general, an animal (preferably a mouse) is immunized with an albuminfusion protein of the invention, or a portion of an albumin fusionprotein of the invention. The splenocytes of such mice are extracted andfused with a suitable myeloma cell line. Any suitable myeloma cell linemay be employed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981)). Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding an albuminfusion protein of the invention, or a portion of an albumin fusionprotein of the invention.

Alternatively, additional antibodies capable of binding to an albuminfusion protein of the invention, or a portion of an albumin fusionprotein of the invention can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to thean albumin fusion protein of the invention (or portion of an albuminfusion protein of the invention)—specific antibody can be blocked by thefusion protein of the invention, or a portion of an albumin fusionprotein of the invention. Such antibodies comprise anti-idiotypicantibodies to the fusion protein of the invention (or portion of analbumin fusion protein of the invention)—specific antibody and are usedto immunize an animal to induce formation of further fusion protein ofthe invention (or portion of an albumin fusion protein of theinvention)—specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed herein. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., InternationalPublication No. WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985)).

b) Isolation of Antibody Fragments Directed Against an Albumin FusionProtein of the Invention, or a Portion of an Albumin Fusion Protein ofthe Invention from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstan albumin fusion protein of the invention, or a portion of an albuminfusion protein of the invention, to which the donor may or may not havebeen exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein byreference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in International Publication No. WO 92/01047. Torescue phase displaying antibody fragments, approximately 10⁹ E. coliharboring the phagemid are used to inoculate 50 ml of 2xTY containing 1%glucose and 100 μg/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D.of 0.8 with shaking. Five ml of this culture is used to inoculate 50 mlof 2xTY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III,see International Publication No. WO 92/01047) are added and the cultureincubated at 37° C. for 45 minutes without shaking and then at 37° C.for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m.for 10 min. and the pellet resuspended in 2 liters of 2xTY containing100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phageare prepared as described in International Publication No. WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phase morphouenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2xTY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 10¹³ transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of an albumin fusion proteinof the invention, or a portion of an albumin fusion protein of theinvention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C.and then washed 3 times in PBS. Approximately 10¹³ TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS. 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of analbumin fusion protein of the invention, or a portion of an albuminfusion protein of the invention, in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., International Publication No. WO 92/01047) and then by sequencing.These ELISA positive clones may also be further characterized bytechniques known in the art, such as, for example, epitope mapping,binding affinity, receptor signal transduction, ability to block orcompetitively inhibit antibody/antigen binding, and competitiveagonistic or antagonistic activity.

Example 17 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing an albumin fusion protein of the present invention, onto apatient. Generally, fibroblasts are obtained from a subject by skinbiopsy. The resulting tissue is placed in tissue-culture medium andseparated into small pieces. Small chunks of the tissue are placed on awet surface of a tissue culture flask, approximately ten pieces areplaced in each flask. The flask is turned upside down, closed tight andleft at room temperature over night. After 24 hours at room temperature,the flask is inverted and the chunks of tissue remain fixed to thebottom of the flask and fresh media (e.g., Ham's F12 media, with 10%FBS, penicillin and streptomycin) is added. The flasks are thenincubated at 37 degree C. for approximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

Polynucleotides encoding an albumin fusion protein of the invention canbe generated using techniques known in the art amplified using PCRprimers which correspond to the 5′ and 3′ end sequences and optionallyhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellstransduced with the vector. The packaging cells now produce infectiousviral particles containing the gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether the albuminfusion protein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 18 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) sequences encoding an albumin fusion protein ofthe invention into an animal. Polynucleotides encoding albumin fusionproteins of the present invention may be operatively linked to (i.e.,associated with) a promoter or any other genetic elements necessary forthe expression of the polypeptide by the target tissue. Such genetherapy and delivery techniques and methods are known in the art, see,for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622,5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479(1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff,Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther.3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996)(incorporated herein by reference).

The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, polynucleotides encoding albumin fusion proteins ofthe present invention may also be delivered in liposome formulations(such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci.772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) whichcan be prepared by methods well known to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Any strongpromoter known to those skilled in the art can be used for driving theexpression of DNA. Unlike other gene therapy techniques, one majoradvantage of introducing naked nucleic acid sequences into target cellsis the transitory nature of the polynucleotide synthesis in the cells.Studies have shown that non-replicating DNA sequences can be introducedinto cells to provide production of the desired polypeptide for periodsof up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within an animal, including muscle, skin, brain, lung, liver,spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage,pancreas, kidney, gall bladder, stomach, intestine, testis, ovary,uterus, rectum, nervous system, eye, gland, and connective tissue.Interstitial space of the tissues comprises the intercellular fluid,mucopolysaccharide matrix among the reticular fibers of organ tissues,elastic fibers in the walls of vessels or chambers, collagen fibers offibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in Ron-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount ofDNA or RNA will be in the range of from about 0.05 g/kg body weight toabout 50 mg/kg, body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of, an aerosolformulation particularly for delivery to lung's or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivois determined as follows. Suitable template DNA for production of mRNAcoding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The template DNA is injected in 0.1 ml of carrier in a 1 ccsyringe through a 27 cause needle over one minute, approximately 0.5 cmfrom the distal insertion site of the muscle into the knee and about 0.2cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for fusion proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of DNA inmuscle following injection may be determined by Southern blot analysisafter preparing total cellular DNA and HIRT supernatants from injectedand control mice. The results of the above experimentation in mice canbe used to extrapolate proper dosages and other treatment parameters inhumans and other animals using naked DNA.

Example 19 Transgenic Animals

The albumin fusion proteins of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express fusion proteins of the invention in humans, as partof a gene therapy protocol.

Any technique known in the art may be used to introduce thepolynucleotides encoding the albumin fusion proteins of the inventioninto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to, pronuclear microinjection(Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carveret al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al.,Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No.4,873,191 (1989)); retrovirus mediated gene transfer into germ lines(Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152(1985)), blastocysts or embryos; gene targeting in embryonic stem cells(Thompson et al., Cell 56:313-321 (1989)); electroporation of cells orembryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction ofthe polynucleotides of the invention using a gene gun (see, e.g., Ulmeret al., Science 259:1745 (1993); introducing nucleic acid constructsinto embryonic pleuripotent stem cells and transferring the stem cellsback into the blastocyst; and sperm-mediated gene transfer (Lavitrano etal., Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol 115:171-229 (1989), whichis incorporated by reference herein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides encoding albumin fusion proteins of theinvention, for example, nuclear transfer into enucleated oocytes ofnuclei from cultured embryonic, fetal, or adult cells induced toquiescence (Campell et al., Nature 380:6466 (1996); Wilmut et al.,Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thepolynucleotides encoding the albumin fusion proteins of the invention inall their cells, as well as animals which carry these polynucleotides insome, but not all their cells, i.e., mosaic animals or chimeric. Thetransgene may be integrated as a single transgene or as multiple copiessuch as in concatamers, e.g., head-to-head tandems or head-to-tailtandems. The transgene may also be selectively introduced into andactivated in a particular cell type by following, for example, theteaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide encoding the fusion protein of theinvention be integrated into the chromosomal site of the endogenous genecorresponding to the Therapeutic protein portion or ablumin portion ofthe fusion protein of the invention, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thepolynucleotide encoding the fsuion protien of the invention has takenplace. The level of mRNA expression of the polynucleotide encoding thefusion protein of the invention in the tissues of the transgenic animalsmay also be assessed using techniques which include, but are not limitedto, Northern blot-analysis of tissue samples obtained from the animal,in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR).Samples of fusion protein-expressing tissue may also be evaluatedimmunocytochemically or immunohistochemically using antibodies specificfor the fusion protein.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene(i.e., polynucleotide encoding an albumin fusion protein of theinvention) on a distinct background that is appropriate for anexperimental model of interest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of fusion proteins of the invention and the Therapeutic proteinand/or albumin component of the fusion protein of the invention,studying conditions and/or disorders associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

Example 20 Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay—Albumin fusion proteins of the invention (includingfusion proteins containing fragments or variants of Therapeutic proteinsand/or albumin or fragments or variants of albumin) can be assessed forits ability to induce activation, proliferation, differentiation orinhibition and/or death in B-cell populations and their precursors. Theactivity of an albumin fusion protein of the invention on purified humantonsillar B cells, measured qualitatively over the dose range from 0.1to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulationassay in which purified tonsillar B cells are cultured in the presenceof either formalin-fixed Staphylococcus aureus Cowan I (SAC) orimmobilized anti-human IgM antibody as the priming agent. Second signalssuch as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicitB cell proliferation as measured by tritiated-thymidine incorporation.Novel synergizing agents can be readily identified using this assay. Theassay involves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD-3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20b pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In vivo Assay—BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of an albumin fusion protein of the invention(including fusion proteins containing fragments or variants ofTherapeutic proteins and/or albumin or fragments or variants ofalbumin). Mice receive this treatment for 4 consecutive days, at whichtime they are sacrificed and various tissues and serum collected foranalyses. Comparison of H&E sections from normal spleens and spleenstreated with the albumin fusion protein of the invention identify theresults of the activity of the fusion protein on spleen cells, such asthe diffusion of peri-arterial lymphatic sheaths, and/or significantincreases in the nucleated cellularity of the red pulp regions, whichmay indicate the activation of the differentiation and proliferation ofB-cell populations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchances to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated with thealbumin fusion protein is used to indicate whether the albumin fusionprotein specifically increases the proportion of ThB+, CD45R(B220) dullB cells over that which is observed in control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andfusion protein treated mice.

The studies described in this example tested activity of fusion proteinsof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of fusion proteins andpolynucleotides of the invention (e.g., gene therapy).

Example 21 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4degrees C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9:5), then washedthree times with PBS. PBMC are isolated by F/H gradient centrifugationfrom human peripheral blood and added to quadruplicate wells(5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS and P/S inthe presence of varying concentrations of an albumin fusion protein ofthe invention (including fusion proteins containing fragments orvariants of Therapeutic proteins and/or albumin or fragments or variantsof albumin) (total volume 200 ul). Relevant protein buffer and mediumalone are controls. After 48 hr. culture at 37 degrees C., plates arespun for 2 min. at 1000 rpm and 100 μl of supernatant is removed andstored −20 degrees C. for measurement of IL-2 (or other cytokines) ifeffect on proliferation is observed. Wells are supplemented with 100 ulof medium containing 0.5 uCi of ³H-thymidine and cultured at 37 degreesC. for 18-24 hr. Wells are harvested and incorporation of ³H-thymidineused as a measure of proliferation. Anti-CD3 alone is the positivecontrol for proliferation. IL-2 (100 U/ml) is also used as a controlwhich enhances proliferation. Control antibody which does not induceproliferation of T cells is used as the negative control for the effectsof fusion proteins of the invention.

The studies described in this example tested activity of fusion proteinsof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of fusion proteins orpolynucleotides of the invention (e.g., gene therapy).

Example 22 Effect of Fusion Proteins of the Invention on the Expressionof MHC Class Ii, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These chances correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of an albumin fusionprotein of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30minutes at 4 degrees C. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (10⁶/ml) are treated with increasing concentrations ofan albumin fusion protein of the invention for 24 hours. LPS (100 ng/ml)is added to the cell culture as positive control. Supernatants from thecell cultures are then collected and analyzed for IL-12 content usingcommercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). Thestandard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inanti-en presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increased expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations of analbumin fusion protein of the invention or LPS (positive control),washed with PBS containing 1% BSA and 0.02 mM sodium azide, and thenincubated with 1:20 dilution of appropriate FITC- or PE-labeledmonoclonal antibodies for 30 minutes at 4 degrees C. After an additionalwash, the labeled cells are analyzed by flow cytometry on a FACScan(Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. Albumin fusion proteins of the invention can be screenedusing the three assays described below. For each of these assays,Peripheral blood mononuclear cells (PBMC) are purified from single donorleukopacks (American Red Cross, Baltimore, Md.) by centrifugationthrough a Histopaque gradient (Sigma). Monocytes are isolated from PBMCby counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated processes (apoptosis). Additionto the culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the fusionprotein to be tested. Cells are suspended at a concentration of 2×10⁶/mlin PBS containing PI at a final concentration of 5 μg/ml, and thenincubated at room temperature for 5 minutes before FACScan analysis. PIuptake has been demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of an albumin fusion protein of theinvention and under the same conditions, but in the absence of thefusion protein. For IL-12 production, the cells are primed overnightwith IFN (100 U/ml) in the presence of the fusion protein. LPS (10ng/ml) is then added. Conditioned media are collected after 24 h andkept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8is then performed using a commercially available ELISA kit (e.g., R & DSystems (Minneapolis, Minn.)) and applying the standard protocolsprovided with the kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×10⁵cell/well. Increasing concentrations of an albumin fusion protein of theinvention are added to the wells in a total volume of 0.2 ml culturemedium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 daysincubation, the plates are centrifuged and the medium is removed fromthe wells. To the macrophage monolayers, 0.2 ml per well of phenol redsolution (140 moral NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, togetherwith the stimulant (200 nM PMA). The plates are incubated at 37° C. for2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well.The absorbance is read at 610 nm. To calculate the amount of H₂O₂produced by the macrophages, a standard curve of a H₂O₂ solution ofknown molarity is performed for each experiment.

The studies described in this example tested activity of fusion proteinsof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of fusion proteins orpolynucleotides of the invention (e.g., gene therapy).

Example 23 Biological Effects of Fusion Proteins of the Invention

Astrocyte and Neuronal Assays

Albumin fusion proteins of the invention can be tested for activity inpromoting the survival, neurite outgrowth, or phenotypic differentiationof cortical neuronal cells and for inducing the proliferation of glialfibrillary acidic protein immunopositive cells, astrocytes. Theselection of cortical cells for the bioassay is based on the prevalentexpression of FGF-1 and FGF-2 in cortical structures and on thepreviously reported enhancement of cortical neuronal survival resultingfrom FGF-2 treatment. A thymidine incorporation assay, for example, canbe used to elucidate an albumin fusion protein of the invention'sactivity on these cells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of an albuminfusion protein of the invention to induce neurite outgrowth can becompared to the response achieved with FGF-2 using, for example, athymidine incorporation assay.

Fibroblast and Endothelial Cell Assays

Human lung, fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test fusionprotein of the invention proteins for 3 days. Alamar Blue (AlamarBiosciences, Sacramento, Calif.) is added to each well to a finalconcentration of 10%. The cells are incubated for 4 hr. Cell viabilityis measured by reading in a CytoFluor fluorescence reader. For the PGF₂assays, the human lung fibroblasts are cultured at 5,000 cells/well in a96-well plate for one day. After a medium change to 0.1% BSA basalmedium, the cells are incubated with FGF-2 or fusion protein of theinvention with or without IL-1α for 24 hours. The supernatants arecollected and assayed for PGE, by ELL kit (Cayman, Ann Arbor, Mich.).For the IL-6 assays, the human lung fibroblasts are cultured at 5,000cells/well in a 96-well plate for one day. After a medium change to 0.1%BSA basal medium, the cells are incubated with FGF-2 or with or withoutan albumin fusion protein of the invention and/or IL-1a for 24 hours.The supernatants are collected and assayed for IL-6 by ELISA kit(Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or an albumin fusionprotein of the invention for 3 days in basal medium before the additionof Alamar Blue to assess effects on growth of the fibroblasts. FGF-2should show a stimulation at 10-2500 ng/ml which can be used to comparestimulation with the fusion protein of the invention.

Cell Proliferation Based on [3H]Thymidine Incorporation

The following [3H]Thymidine incorporation assay can be used to measurethe effect of a Therapeutic proteins, e.g., growth factor proteins, onthe proliferation of cells such as fibroblast cells, epithelial cells orimmature muscle cells.

Sub-confluent cultures are arrested in G1 phase by an 18 h incubation inserum-free medium. Therapeutic proteins are then added for 24 h andduring the last 4 h, the cultures are labeled with [3H]thymidine, at afinal concentration of 0.33 μM (25 Ci/mmol, Amersham, Arlington Heights,Ill.). The incorporated [3H]thymidine is precipitated with ice-cold 10%trichloroacetic acid for 24 h. Subsequently, the cells are rinsedsequentially with ice-cold 10% trichloroacetic acid and then withice-cold water. Following lysis in 0.5 M NaOH, the lysates and PBSrinses (500 ml) are pooled, and the amount of radioactivity is measured.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released.Subsequently, MPP⁻ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, an albumin fusion protein of the inventioncan be evaluated to determine whether it has an action similar to thatof FGF-2 in enhancing dopaminergic neuronal survival in vitro and it canalso be tested in vivo for protection of dopaminergic neurons in thestriatum from the damage associated with MPTP treatment. The potentialeffect of an albumin fusion protein of the invention is first examinedin vitro in a dopaminergic neuronal cell culture paradigm. The culturesare prepared by dissecting the midbrain floor plate from gestation day14 Wistar rat embryos. The tissue is dissociated with trypsin and seededat a density of 200,000 cells/cm² on polyorthinine-laminin coated glasscoverslips. The cells are maintained in Dulbecco's Modified Eagle'smedium and F12 medium containing hormonal supplements (N1). The culturesare fixed with paraformaldehyde after 8 days in vitro and are processedfor tyrosine hydroxylase, a specific marker for dopaminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a Therapeutic protein acts to prolong the survival ofdopaminergic neurons, it would suggest that the fusion protein may beinvolved in Parkinson's Disease.

The studies described in this example tested activity of albumin fusionproteins of the invention. However, one skilled in the art could easilymodify the exemplified studies to test the activity of fusion proteinsand polynucleotides of the invention (e.g., gene therapy).

Example 24 The Effect of Albumin Fusion Proteins of the Invention on theGrowth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. An albuminfusion protein of the invention, and positive controls, such as VEGF andbasic FGF (bFGF) are added, at varying concentrations. On days 4and 6,the medium is replaced. On day 8, cell number is determined with aCoulter Counter.

An increase in the number of HUVEC cells indicates that the fusionprotein may proliferate vascular endothelial cells, while a decrease inthe number of HUVEC cells indicates that the fusion protein inhibitsvascular endothelial cells.

The studies described in this example tested activity of an albuminfusion protein of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of a fusionprotiem and polynucleotides of the invention.

Example 25 Rat Corneal Wound Healing Model

This animal model shows the effect of an albumin fusion protein of theinvention on neovascularization. The experimental protocol includes:

Making a 1-1.5 mm long incision from the center of cornea into thestromal layer.

Inserting a spatula below the lip of the incision facing the outercorner of the eye. Making a pocket (its base is 1-1.5 mm form the edgeof the eye).

Positioning a pellet, containing 50 ng-5 ug of an albumin fusion proteinof the invention, within the pocket.

Treatment with an an albumin fusion protein of the invention can also beapplied topically to the corneal wounds in a dosage range of 20 mg-500mg (daily treatment for five days).

The studies described in this example test the activity of an albuminfusion protein of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of fusionproteins and polynucleotides of the invention (e.g., gene therapy).

Example 26 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

Diabetic db+/db+ Mouse Model.

To demonstrate that an albumin fusion protein of the inventionaccelerates the healing process, the genetically diabetic mouse model ofwound healing is used. The full thickness wound healing model in thedb+/db+ mouse is a well characterized, clinically relevant andreproducible model of impaired wound healing. Healing of the diabeticwound is dependent on formation of granulation tissue andre-epithelialization rather than contraction (Gartner, M. H. et al., J.Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol.136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

An albumin fusion protein of the invention is administered usina at arange different doses, from 4 mg to 500 mg per wound-per day for 8 daysin vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3)treated group.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment. with an albumin fusion protein of the invention. Thisassessment included verification of the presence of cell accumulation,inflammatory cells, capillaries, fibroblasts, re-epithelialization andepidermal maturity (Greenhalgrh, D. G. et al., Am. J. Pathol. 136:1235(1990)). A calibrated lens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting, intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, Glucocorticoids and Woundhealing. In: Anti-inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl et al., J. Immunol. 115: 476-481 (1975);Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retardwound healing by inhibiting angiogenesis, decreasing vascularpermeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)),fibroblast proliferation, and collagen synthesis (Beck et al., GrowthFactors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797(1978)) and producing a transient reduction of circulating monocytes(Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl,“Glucocorticoids and wound healing”, In: Antiinflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al.,Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In:Antiinflammatory Steroid Action: Basic and Clinical Aspects, AcademicPress, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad.Sci. USA 86: 2229-2233 (1989)).

To demonstrate that an albumin fusion protein of the invention canaccelerate the healing process, the effects of multiple topicalapplications of the fusion protein on full thickness excisional skinwounds in rats in which healing has been impaired by the systemicadministration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to that described above. Onthe day of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

The fusion protein of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Three groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm², the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith an albumin fusion protein of the invention. A calibrated lensmicrometer is used by a blinded observer to determine the distance ofthe wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example tested activity of an albuminfusion protein of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of fusionproteins and polynucleotides of the invention (e.g., gene therapy).

Example 27 Lymphedema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects ofan albumin fusion protein of the invention in lymphangiogenesis- andre-establishment of the lymphatic circulatory system in the rat hindlimb. Effectiveness is measured by swelling volume of the affected limb,quantification of the amount of lymphatic vasculature, total bloodplasma protein, and histopathology. Acute lymphedema is observed for7-10 days. Perhaps more importantly, the chronic progress of the edemais followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats-weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements: are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially-allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated. The 2 proximal and 2 distal lymphatic vessels and distal bloodsupply of the popliteal node are then ligated by suturing. The popliteallymph node, and any accompanying adipose tissue, is then removed bycutting connective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin-flaps are sealed byusing liquid skin (Vetbond) (A J Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effect ofplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople and those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), and both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software (Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2⁺ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs are amputated using a quillitine, then bothexperimental and control legs are cut at the ligature and weighed. Asecond weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80EC until sectioning. Upon sectioning the muscle is observedunder fluorescent microscopy for lymphatics.

The studies described in this example tested activity of fusion proteinsof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of fusion protein andpolynucleotides of the invention (e.g., gene therapy).

Example 28 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby an Albumin Fusion Protein of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-α), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of an albumin fusion protein of the invention to mediate asuppression of TNF-a induced CAM expression can be examined. A modifiedELISA assay which uses ECs as a solid phase absorbent is employed tomeasure the amount of CAM expression on TNF-a treated ECs whenco-stimulated with a member of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37 degree C. humidified incubatorcontaining 5% CO₂. HUVECs are seeded in 96-well plates at concentrationsof 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37 degree C. Following incubation, thecells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37 degree C. for either 5 h(selectin and integrin expression) or 24 h (integrin expression only).Plates are aspirated to remove medium and 100 μl of 0.1%paraformaldehyde-PBS(with Ca++ and Mga++) is added to each well. Platesare held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1X withPBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000dilution). to each well and incubated at 37° C. for 30 min. Wells arewashed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol PhosphatepNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPPsubstrate in glycine buffer is added to each test well. Standard wellsin triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000(10⁰)>10^(−0.5)>10⁻¹>10^(−1.5). 5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added toeach of the standard wells. The plate must be incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

The studies described in this example tested activity of fusion proteinsof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of fusion proteins andpolynucleotides of the invention (e.g., gene therapy).

Example 29 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation andproliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factorscalled Signal Transducers and Activators of Transcription, or “STATs.”There are six members of the STATs family. Stat1 and Stat3 are presentin many cell types, as is State (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleusupon tyrosine phosphorylation by a set of kinases known as the JanusKinase (“Jaks”) family. Jaks represent a distinct family of solubletyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinasesdisplay significant sequence similarity and are generally catalyticallyinactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in theTable below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995)). A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-α, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proximal region encodingTrp-Ser-Xaa-Trp-Ser (SEQ ID NO: 37)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which inturn activate STATs, which then translocate and bind to GAS elements.This entire process is encompassed in the Jaks-STATs signal transductionpathway. Therefore, activation of the Jaks-STATs pathway, reflected bythe binding of the GAS or the ISRE element, can be used to indicateproteins involved in the proliferation and differentiation of cells. Forexample, growth factors and cytokines are known to activate theJaks-STATs pathway (See Table below). Thus, by using GAS elements linkedto reporter molecules, activators of the Jaks-STATs pathway can beidentified. JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) or ISREIFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 >IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotropic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP) Il-11(Pleiotropic) ? + ? ? 1, 3 OnM(Pleiotropic)? + + ? 1, 3 LIF(Pleiotropic) ? + + ? 1, 3 CNTF(Pleiotropic) −/+ + + ?1, 3 G-CSF(Pleiotropic) ? + ? ? 1, 3 IL-12(Pleiotropic) + − + + 1, 3 g-Cfamily IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid) − +− + 6 GAS(IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9(lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? +? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6)IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5GAS(B-CAS > IRF1 = IFP > Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is usedin the Biological Assays described in Examples 32-33, a PCR basedstrategy is employed to generate a GAS-SV40 promoter sequence. The 5′primer contains four tandem copies of the GAS binding site found in theIRF1 promoter and previously demonstrated to bind STATs upon inductionwith a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).),although other GAS or ISRE elements can be used instead. The 5′ primeralso contains 18 bp of sequence complementary to the SV40 early promotersequence and is flanked with an XhoI site. The sequence of the 5′ primeris: (SEQ ID NO: 38) 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the SV40 promoter and isflanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ IDNO: 39)

PCR amplification is performed using the SV40 promoter template presentin the B-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI/Hind III and subcloned into BLSK2.(Stratagene.) Sequencing with forward and reverse primers confirms thatthe insert contains the following sequence: (SEQ ID NO: 40)5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2reporter construct is next engineered. Here, the reporter molecule is asecreted alkaline phosphatase, or “SEAP.” Clearly, however, any reportermolecule can be instead of SEAP, in this or in any of the otherExamples. Well known reporter molecules that can be used instead of SEAPinclude chloramphenicol acetyltransferase (CAT), luciferase, alkalinephosphatase, B-galactosidase, green fluorescent protein (GCFP), or anyprotein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element issubcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing theGAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAPvector using SaIlI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 32-33.

Other constructs can be made using the above description and replacingGAS with a different promoter sequence. For example, construction ofreporter molecules containing EGR and NF-KB promoter sequences aredescribed in Examples 34 and 35. However, many other promoters can besubstituted using the protocols described in these Examples. Forinstance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted,alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, I1-2/NFAT, orNF-KB/GAS). Similarly, other cell lines can be used to test reporterconstruct activity, such as HELA (epithelial), HUVEC (endothelial), Reh(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 30 Assay for SEAP Activity

As a reporter molecule for the assays described in examples disclosedherein, SEAP activity is assayed using the Tropix Phospho-light Kit(Cat. BP-400) according to the following general procedure. The TropixPhospho-light Kit supplies the Dilution, Assay, and Reaction Buffersused below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of2.5× dilution buffer into Optiplates containing 35 ul of a solutioncontaining an albumin fusion protein of the invention. Seal the plateswith a plastic sealer and incubate at 65 degree C. for 30 min. Separatethe Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenserand prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate atroom temperature 5 min., Empty the dispenser and prime with the ReactionBuffer (see the Table below). Add 50 ul Reaction Buffer and incubate atroom temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on a luminometer, thus one should treat 5 plates ateach time and start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, andprint the results. An increase in chemiluminescence indicates reporteractivity. Reaction Buffer Formulation: # of plates Rxn buffer diluent(ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 854.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.529 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 1859.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 21510.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 24512.25 48 250 12.5 49 255 12.75 50 260 13

Example 31 Assay Identifying Neuronal Activity

When cells undergo differentiation and proliferation, a group of genesare activated through many different signal transduction pathways. Oneof these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, the ability of fusion proteins of the invention toactivate cells can be assessed.

Particularly, the following protocol is used to assess neuronal activityin PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are knownto proliferate and/or differentiate by activation with a number ofmitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growthfactor), and EGF (epidermal growth factor). The EGR1 gene expression isactivated during this treatment. Thus, by stably transfecting PC12 cellswith a construct containing an EGR promoter linked to SEAP reporter,activation of PC12 cells by an albumin fusion protein of the presentinvention can be assessed.

The EGR/SE-P reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers: (SEQ ID NO: 41)5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO: 42)5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′

Using the GAS:SEAP/Neo vector produced in Example 29, EGR1 amplifiedproduct can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5%heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/mlpenicillin and 100 ug/ml streptomycin on a precoated 10 cm tissueculture dish. One to four split is done every three to four days. Cellsare removed from the plates by scraping and resuspended with pipettingup and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC12 using techniques known inthe art. EGR-SEAP/PC12 stable cells are obtained by growing the cells in300 ug/ml G418. The G418-free medium is used for routine growth butevery one to two months, the cells should be re-grown in 300 ug/ml G418for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to80% confluent is screened by removing the old medium. Wash the cellsonce with PBS (Phosphate buffered saline). Then starve the cells in lowserum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS withantibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrapeoff the cells from the plate, suspend the cells well in 2 ml low serummedium. Count the cell number and add more low serum medium to reachfinal cell density as 5×10⁵ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add a series of differentconcentrations of an albumin fusion protein of the inventon, 37 degreeC. for 48 to 72 hr. As a positive control, a growth factor known toactivate PC12 cells through EGR can be used, such as 50 ng/ul ofNeuronal Growth Factor (NGF). Over fifty-fold induction of SEAP istypically seen in the positive control wells. SEAP assay may beroutinely performed using techniques known in the art and/or asdescribed in Example 30.

Example 32 Assay for T-Cell Activity

The following protocol is used to assess T-cell activity by identifyingfactors, and determining whether an albumin fusion protein of theinvention proliferates and/or differentiates T-cells. T-cell activity isassessed using the GAS/SEAP/Neo construct produced in Example 29. Thus,factors that increase SEAP activity indicate the ability to activate theJaks-STATS signal transduction pathway. The T-cell used in this assay isJurkat T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCCAccession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582)cells can also be used.

Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order togenerate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75wells containing 200 ul of cells. Thus, it is either scaled up, orperformed in multiple to generate sufficient cells for multiple 96 wellplates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep.Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmidDNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C andincubate at room temperature for 1545 mins.

During the incubation period, count cell concentration, spin down therequired number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degree C. for 6hrs. After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10%serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated withvarying concentrations of one or more fusion proteins of the presentinvention.

On the day of treatment with the fusion protein, the cells should bewashed and resuspended in fresh RPMI+10% serum to a density of 500,000cells per ml. The exact number of cells required will depend on thenumber of fusion proteins and the number of different concentrations offusion proteins being screened. For one 96 well plate, approximately 10million cells (for 10 plates, 100 million cells) are required.

The well dishes containing Jurkat cells treated with the fusion proteinare placed in an incubator for 48 hrs (note: this time is variablebetween 48-72 hrs). 35 ul samples from each well are then transferred toan opaque 96 well plate using a 12 channel pipette. The opaque platesshould be covered (using sellophene covers) and stored at −20 degree C.until SEAP assays are performed according to Example 30. The platescontaining the remaining treated cells are placed at 4 degree C. andserve as a source of material for repeating the assay on a specific wellif desired.

As a positive control, 100 Unit/ml interferon: gamma can be used whichis known to activate Jurkat T cells. Over 30 fold induction is typicallyobserved in the positive control wells.

The above protocol may be used in the generation of both transient, aswell as, stable transfected cells, which would be apparent to those ofskill in the art.

Example 33 Assay for T-cell Activity

NF-KB (Nuclear Factor KB) is a transcription factor activated by a widevariety of agents including the inflammatory cytokines IL-1 and TNF,CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure toLPS or thrombin, and by expression of certain viral gene products. As atranscription factor, NF-KB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-KB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

In non-stimulated conditions, NF-KB is retained in the cytoplasm withI-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylatedand degraded, causing NF-KB to shuttle to the nucleus, therebyactivating transcription of target genes. Target genes activated byNF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

Due to its central role and ability to respond to a range of stimuli,reporter constructs utilizing the NF-KB promoter element are used toscreen the fusion protein. Activators or inhibitors of NF-KB would beuseful in treating, preventing, and/or diagnosing, diseases. Forexample, inhibitors of NF-KB could be used to treat those diseasesrelated to the acute or chronic activation of NF-KB, such as rheumatoidarthritis.

To construct a vector containing the NF-KB promoter element, a PCR basedstrategy is employed. The upstream primer contains four tandem copies ofthe NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO: 43), 18 bp of sequencecomplementary to the 5′ end of the SV40 early promoter sequence, and isflanked with an XhoI site: (SEQ ID NO: 44)5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site: (SEQ ID NO: 39)5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′

PCR amplification is performed using the SV40 promoter template presentin the pB-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI and Hind III and subcloned into BLSK2-.(Stratagene) Sequencing with the T7 and T3 primers confirms the insertcontains the following sequence: (SEQ ID NO: 45)5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3′

Next, replace the SV40 minimal promoter element present in thepSEAlr—promoter plasmid (Clontech) with this NF-KB/SV40 fragment using,XhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAPcassette is removed from the above NF-KB/SEAP vector using restrictionenzymes SalI and NotI, and inserted into a vector containing neomycinresistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted intopGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 withSalI and NotI.

Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells arecreated and maintained according to the protocol described in Example32. Similarly, the method for assaying fusion proteins with these stableJurkat T-cells is also described in Example 32. As a positive control,exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10, and H11,with a 5-10 fold activation typically observed.

Example 33 Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity of an albuminfusion protein of the present invention by determining whether thefusion protein proliferates and/or differentiates myeloid cells. Myeloidcell activity is assessed using the GAS/SE-P/Neo construct produced inExample 29. Thus, factors that increase SEAP activity indicate theability to activate the Jaks-STATS signal transduction pathway. Themyeloid cell used in this assay is U937, a pre-monocyte cell line,although TF-1, HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo constructproduced in Example 29, a DEAE-Dextran method (Kharbanda et. at., 1994,Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10⁷U937 cells and wash with PBS. The U937 cells are usually grown in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS)supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM NaCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degrees C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degree C. for 36hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400ug/ml G418. The G418-free medium is used for routine growth but everyone to two months, the cells should be re-grown in 400 ug/ml G418 forcouple of passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten96-well plates assay) and wash with PBS. Suspend the cells in 200 mlabove described growth medium, with a final density of 5×10⁵ cells/ml.Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add different concentrations of the fusion protein. Incubate at 37 degeeC for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gammacan be used which is known to activate U937 cells. Over 30 foldinduction is typically observed in the positive control wells. SEAPassay the supernatant according, to methods known in the art and/or theprotocol described in Example 30.

Example 34 Assay Identifying Changes in Small Molecule Concentration andMembrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levelsof small molecules, such as calcium, potassium, sodium, and pH, as wellas alter membrane potential. These alterations can be measured in anassay to identify fusion proteins which bind to receptors of aparticular cell. Although the following protocol describes an assay forcalcium, this protocol can easily be modified to detect changes inpotassium, sodium, pH, membrane potential, or any other small moleculewhich is detectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) tomeasure changes in fluorescent molecules (Molecular Probes) that bindsmall molecules. Clearly, any fluorescent molecule detecting a smallmolecule can be used instead of the calcium fluorescent molecule, fluo-4(Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000 cells/well in a Co-starblack 96-well plate with clear bottom. The plate is incubated in a CO₂incubator for 20 hours. The adherent cells are washed two times inBiotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. Toload the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to eachwell. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60min. The plate is washed four times in the Biotek washer with HBSSleaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media.Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conicaltube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is addedto each ml of cell suspension. The tube is then placed in a 37 decrees Cwater bath for 30-60 min. The cells are washed twice with HBSS,resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate isthen washed once in Denley Cell Wash with 200 ul; followed by anaspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule,such as fluo-4. The fusion protein of the invention is added to thewell, and a chance in fluorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is setfor the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventcaused by an albumin fusion protein of the present invention or amolecule induced by an albumin fusion protein of the present invention,which has resulted in an increase in the intracellular Ca⁺⁺concentration.

Example 35 Assay Identifying Tyrosine Kinase Activity

The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase (RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, identifying whether an albumin fusion proteinof the present invention or a molecule induced by a fusion proetin ofthe present invention is capable of activating tyrosine kinase signaltransduction pathways is of interest. Therefore, the following protocolis designed to identify such molecules capable of activating thetyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalgre Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes ofLoprodyne plates (20,000/200 ml/well) and cultured overnight in completemedium. Cells are quiesced by incubation in serum-free basal medium for24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or a differentconcentrations of an albumin fusion protein of the invention, the mediumwas removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 MNaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and acocktail of protease inhibitors (# 1836170) obtained from BoeheringerMannheim (Indianapolis, Ind.)) is added to each well and the plate isshaken on a rotating shaker for 5 minutes at 4° C. The plate is thenplaced in a vacuum transfer manifold and the extract filtered throughthe 0.45 mm membrane bottoms of each well using house vacuum. Extractsare collected in a 96-well catch/assay plate in the bottom of the vacuummanifold and immediately placed on ice. To obtain extracts clarified bycentrifugation, the content of each well, after detergent solubilizationfor 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C.at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity.Although many methods of detecting tyrosine kinase activity are known,one method is described here.

Generally, the tyrosine kinase activity of an albumin fusion protein ofthe invention is evaluated by determining its ability to phosphorylate atyrosine residue on a specific substrate (a biotinylated peptide).Biotinylated peptides that can be used for this purpose include PSK1(corresponding to amino acids 6-20 of the cell division kinase cdc2-p34)and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptidesare substrates for a range of tyrosine kinases and are available fromBoehringer Mannheim.

The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂ 0.5 mg/ml BSA), then 5 ul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degree C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot ofreaction mixture to a microtiter plate (MTP) module and incubating at 37degree C. for 20 min. This allows the streptavidin coated 96 well plateto associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti-phospotyrosineantibody conjugated to horse radish peroxidase (anti-P-Tyr-POD(0.5u/ml))to each well and incubate at 37 degree C. for one hour. Wash the well asabove.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim)and incubate at room temperature for at least 5 mins (up to 30 min).Measure the absorbance of the sample at 405 nm by using ELISA reader.The level of bound peroxidase activity is quantitated using an ELISAreader and reflects the level of tyrosine kinase activity.

Example 36 Assay Identifying Phosphorylation Activity

As a potential alternative and/or complement to the assay of proteintyrosine kinase activity described in Example 35, an assay which detectsactivation (phosphorylation) of major intracellular signal transductionintermediates can also be used. For example, as described below oneparticular assay can detect tyrosine phosphorylation of the Erk-1 andErk-2 kinases. However, phosphorylation of other molecules, such as Raf,JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specifickinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,phosphotyrosine, or phosphothreonine molecule, can be detected bysubstituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-wellELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp,(RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBSfor 1 hr at RT. The protein G plates are then treated with 2 commercialmonoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT)(Santa Cruz Biotechnology). (To detect other molecules, this step caneasily be modified by substituting a monoclonal antibody detecting anyof the above described molecules.) After 3-5 rinses with PBS, the platesare stored at 4 degree C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplateand cultured overnight in growth medium. The cells are then starved for48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) orvarying concentrations of the fusion protein of the invention for 5-20minutes. The cells are then solubilized and extracts filtered directlyinto the assay plate.

After incubation with the extract for 1 hr at RT, the wells are againrinsed. As a positive control, a commercial preparation of MAP kinase(10 ng/well) is used in place of A431 extract. Plates are then treatedwith a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation by the fusion protein of thepresent invention or a molecule induced by an albumin fusion protein ofthe present invention.

Example 37 Assay for the Stimulation of Bone Marrow CD34+ CellProliferation

This assay is based on the ability of human CD34+ to proliferate in thepresence of hematopoietic growth factors and evaluates the ability offusion proteins of the inventon to stimulate proliferation of CD34+cells.

It has been previously shown that most mature precursors will respond toonly a single signal. More immature precursors require at least twosignals to respond. Therefore, to test the effect of fusion proteins ofthe invention on hematopoietic activity of a wide range of progenitorcells, the assay contains a given fusion protein of the invention in thepresence or absence of hematopoietic growth factors. Isolated cells arecultured for 5 days in the presence of Stem Cell Factor (SCF) incombination with tested sample. SCF alone has a very limited effect onthe proliferation of bone marrow (BM) cells, acting in such conditionsonly as a “survival” factor. However, combined with any factorexhibiting stimulatory effect on these cells (e.g., IL-3), SCF willcause a synergistic effect. Therefore, if the tested fusion protein hasa stimulatory effect on hematopoietic progenitors, such activity can beeasily detected. Since normal BM cells have a low level of cyclingcells, it is likely that any inhibitory effect of a given fusion proteinmight not be detected. Accordingly, assays for an inhibitory effect onprogenitors is preferably tested in cells that are first subjected to invitro stimulation with SCF+IL+3, and then contacted with the compoundthat is being evaluated for inhibition of such induced proliferation.

Briefly, CD34+ cells are isolated using methods known in the art. Thecells are thawed and resuspended in medium (QBSF 60 serum-free mediumwith 1% L-glutamine (500 ml) Quality Biological, Inc., Gaithersburg, Md.Cat# 160-204-101). After several gentle centrifugation steps at 200×g,cells are allowed to rest for one hour. The cell count is adjusted to2.5×10⁵ cells/ml. During this time, 100 μl of sterile water is added tothe peripheral wells of a 96-well plate. The cytokines that can betested with an albumin fusion protein of the invention in this assay isrhSCF (R&D Systems, Minneapolis, Minn., Cat# 255-SC) at 50 ng/ml aloneand in combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis,Minn., Cat# 203-ML) at 30 ng/ml. After one hour, 10 μl of preparedcytokines, varying concentrations of an albumin fusion protein of theinvention, and 20 μl of diluted cells are added to the media which isalready present in the wells to allow for a final total volume of 100μl. The plates are then placed in a 37° C./5% CO₂ incubator for fivedays.

Eighteen hours before the assay is harvested, 0.5 μCi/well of [3H]Thymidine is added in a 10 μl volume to each well to determine theproliferation rate. The experiment is terminated by harvesting the cellsfrom each 96-well plate to a filtermat using the Tomtec Harvester 96.After harvesting, the filtermats are dried, trimmed and placed intoOmniFilter assemblies consisting of one OmniFilter plate and oneOmniFilter Tray. 60 μl Microscint is added to each well and the platesealed with TopSeal-A press-on sealing film A bar code 15 sticker isaffixed to the first plate for counting. The sealed plates are thenloaded and the level of radioactivity determined via the Packard TopCount and the printed data collected for analysis. The level ofradioactivity reflects the amount of cell proliferation.

The studies described in this example test the activity of a givenfusion protein to stimulate bone marrow CD34+ cell proliferation. Oneskilled in the art could easily modify the exemplified studies to testthe activity of fusion porteins and polynucleotides of the invention(e.g., gene therapy) as well as agonists and antagonists thereof. Theability of an albumin fusion protein of the invention to stimulate theproliferation of bone marrow CD34+ cells indicates that the albuminfusion protein and/or polynucleotides corresponding to the fusionprotein are useful for the diagnosis and treatment of disordersaffecting the immune system and hematopoiesis. Representative uses aredescribed in the “Immune Activity” and “Infectious Disease” sectionsabove, and elsewhere herein.

Example 38 Assay for Extracellular Matrix Enhanced Cell Response(EMECR),

The objective of the Extracellular Matrix Enhanced Cell Response (EMECR)assay is to evaluate the ability of fusion proteins of the invention toact on hematopoietic stem cells in the context of the extracellularmatrix (ECM) induced signal.

Cells respond to the regulatory factors in the context of signal(s)received from the surrounding microenvironment. For example,fibroblasts, and endothelial and epithelial stem cells fail to replicatein the absence of signals from the ECM. Hematopoietic stem cells canundergo self-renewal in the bone marrow, but not in in vitro suspensionculture. The ability of stem cells to undergo self-renewal in vitro isdependent upon their interaction with the stromal cells and the ECMprotein fibronectin (fn). Adhesion of cells to fn is mediated by theα₅.β₁ and α₄.β₁ integrin receptors, which are expressed by human andmouse hematopoietic stem cells. The factor(s) which integrate with theECM environment and are responsible for stimulating stem cellself-renewal havea not yet been identified. Discovery of such factorsshould be of great interest in gene therapy and bone marrow transplantapplications Briefly, polystyrene, non tissue culture treated, 96-wellplates are coated with fn fragment at a coating concentration of 0.2μg/cm². Mouse bone marrow cells are plated (1,000 cells/well) in 0.2 mlof serum-free medium. Cells cultured in the presence of IL-3 (5ng/ml)+SCF (50 ng/ml) would serve as the positive control, conditionsunder which little self-renewal but pronounced differentiation of thestem cells is to be expected. Albumin fusion proteins of the inventionare tested with appropriate negative controls in the presence andabsence of SCF(5.0 ng/ml), where volume of the administed compositioncontaining the albumin fusion protein of the invention represents 10% ofthe total assay volume. The plated cells are then allowed to grow byincubating in a low oxygen environment (5% CO₂, 7% O₂, and 88% N₂)tissue culture incubator for 7 days. The number of proliferating cellswithin the wells is then quantitated by measuring thymidineincorporation into cellular DNA. Verification of the positive hits inthe assay will require phenotypic characterization of the cells, whichcan be accomplished by scaling up of the culture system and usingappropriate antibody reagents against cell surface antigens and FACScan.

One skilled in the art could easily modify the exemplified studies totest the activity of albumin fusion proteins and polynucleotides of theinvention (e.g., grene therapy).

If a particular fusion protein of the present invention is found to be astimulator of hematopoietic progenitors, the fusion protein andpolynucleotides corresponding to the fusion protein may be useful forexample, in the diagnosis and treatment of disorders affecting theimmune system and hematopoiesis. Representative uses are described inthe “Immune Activity” and “Infectious Disease” sections above, andelsewhere herein. The fusion protein may also be useful in the expansionof stem cells and committed progenitors of various blood lineages, andin the differentiation and/or proliferation of various cell types.

Additionally, the albumin fusion proteins of the invention andpolynucleotides encoding albumin fusion proteins of the invention, mayalso be employed to inhibit the proliferation and differentiation ofhematopoietic cells and therefore may be employed to protect bone marrowstem cells from chemotherapeutic agents during chemotherapy. Thisantiproliferative effect may allow administration of higher doses ofchemotherapeutic agents and, therefore, more effective chemotherapeutictreatment.

Moreover, fusion proteins of the invention and polynucleotides encodingalbumin fusion proteins of the invention may also be useful for thetreatment and diagnosis of hematopoietic related disorders such as,anemnia, pancytopenia, leukopenia, thrombocytopenia or leukemia, sincestromal cells are important in the production of cells of hematopoieticlineages. The uses include bone marrow cell ex-vivo culture, bone marrowtransplantation, bone marrow reconstitution, radiotherapy orchemotherapy of neoplasia.

Example 39 Human Dermal Fibroblast and Aortic Smooth Muscle CellProliferation

An albumin fusion protein of the invention is added to cultures ofnormal human dermal fibroblasts (NHDF) and human aortic smooth musclecells (AoSMC) and two co assays are performed with each sample. Thefirst assay examines the effect of the fusion protein on theproliferation of normal human dermal fibroblasts (NHDF) or aortic smoothmuscle cells (AoSMC). Aberrant growth of fibroblasts or smooth musclecells is a part of several pathological processes, including fibrosis,and restenosis. The second assay examines IL6 production by both NHDFand SMC. IL6 production is an indication of functional activation.Activated cells will have increased production of a number of cytokinesand other factors, which can result in a proinflammatory orimmunomodulatory outcome. Assays are run with and without co-TNFastimulation, in order to check for costimulatory or inhibitory activity.

Briefly, on day 1, 96-well black plates are set tip with 1000 cells/well(NHDF) or 2000 cells/well (AoSMC) in 100 μl culture media. NHDF culturemedia contains: Clonetics FB basal media, 1 mg/ml hFGF, 5 mg/ml insulin,50 mg/ml gentamycin, 2% FBS, while AoSMC culture media containsClonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF,50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5% FBS. After incubationat 37° C. for at least 4-5 hours culture media is aspirated and replacedwith growth arrest media. Growth arrest media for NHDF containsfibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth arrestmedia for AoSMC contains SM basal media, 50 mg/ml gentamycin, 50 μg/mlAmphotericin B, 0.4% FBS. Incubate at 37° C. until day 2.

On day 2, serial dilutions and templates of an albumin fusion protein ofthe invention are designed such that they always include media controlsand known-protein controls. For both stimulation and inhibitionexperiments, proteins are diluted in growth arrest media. For inhibitionexperiments, TNFa is added to a final concentration of 2 ng/ml (NHDF) or5 ng/ml (AoSMC). Add ⅓ vol media containing controls or an albuminfusion protein of the invention and incubate at 37 degrees C./5% CO₂until day 5.

Transfer 60 μl from each well to another labeled 96-well plate, coverwith a plate-sealer, and store at 4 degrees C until Day 6 (for IL6ELISA). To the remaining 100 μl in the cell culture plate, asepticallyadd Alamar Blue in an amount equal to 10% of the culture volume (10 μl).Return plates to incubator for 3 to 4 hours. Then measure fluorescencewith excitation at 530 nm and emission at 590 nm using the CytoFluor.This yields the growth stimulation/inhibition data.

On day 5, the IL6 ELISA is performed by coating a 96 well plate with50-100 ul/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH7.4, incubate ON at room temperature.

On day 6, empty the plates into the sink and blot on paper towels.Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with200 ul/well of Pierce Super Block blocking buffer in PBS for 1-2 hr andthen wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates onpaper towels. Then add 50 μl/well of diluted Anti-Human IL-6 Monoclonal,Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock inmedia (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row ofplate. Cover the plates and incubate for 2 hours at RT on shaker.

Plates are washed with wash buffer and blotted on paper towels. DiluteEU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 μl/well.Cover the plate and incubate 1 h at RT. Plates are again washed withwash buffer and blotted on paper towels.

Add 100 μl/well of Enhancement Solution. Shake for 5 minutes. Read theplate on the Wallac DELFIA Fluorometer. Readings from triplicate samplesin each assay were tabulated and averaged.

A positive result in this assay suggests AoSMC cell proliferation andthat the albumin fusion protein may be involved in dermal fibroblastproliferation and/or smooth muscle cell proliferation. A positive resultalso suggests many potential uses of the fusion protein andpolynucleotides encoding the albumin fusion protein. For example,inflammation and immune responses, wound healing, and angiogenesis, asdetailed throughout this specification. Particularly, fusion proteinsmay be used in wound healing and dermal regeneration, as well as thepromotion of vasculogenesis, both of the blood vessels and lymphatics.The growth of vessels can be used in the treatment of, for example,cardiovascular diseases. Additionally, fusion proteins showingantagonistic activity in this assay may be useful in treating diseases,disorders, and/or conditions which involve angiogenesis by acting as ananti-vascular agent (e.g., anti-angiogenesis). These diseases,disorders, and/or conditions are known in the art and/or are describedherein, such as, for example, malignancies, solid tumors, benign tumors,for example hemangiomas, acoustic neuromas, neurofibromas, trachomas,and pyogenic granulomas; artheroscleric plaques; ocular angiogenicdiseases, for example, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma,retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia(abnormal blood vessel growth) of the eye; rheumatoid arthritis;psoriasis; delayed wound healing; endometriosis; vasculogenesis;granulations; hypertrophic scars (keloids); nonunion fractures;scieroderma; trachoma; vascular adhesions; myocardial angiogenesis;coronary collaterals; cerebral collaterals; arteriovenous malformations;ischemic limb angiogenesis; Osler-Webber Syndrome; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;fibromuscular dysplasia; wound granulation; Crohn's disease; andatherosclerosis. Moreover, albumin fusion proteins that act asantagonists in this assay may be useful in treatinganti-hyperproliferative diseases and/or anti-inflammatory known in theart and/or described herein.

Example 40 Cellular Adhesion Molecule (CAM) Expression on EndothelialCells

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, follows.a multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells(HUVECs)) are grown in a standard 96 well plate to confluence, growthmedium is removed from the cells and replaced with 100 μl of 199 Medium(10% fetal bovine serum (FBS)). Samples for testing (containing analbumin fusion protein of the invention) and positive or negativecontrols are added to the plate in triplicate (in 10 μl volumes). Platesare then incubated at 37° C. for either 5 h (selectin and integrinexpression) or 24 h (integrin expression only). Plates are aspirated toremove medium and 100 μl of 0.1% paraformaldehyde-PBS(with Ca++ andMg++) is added to each well. Plates are held at 4° C. for 30 min.Fixative is removed from the wells and wells are washed 1× withPBS(+Ca,Mg)+0.5% BSA and drained. 10 μl of diluted primary antibody isadded to the test and control wells. Anti-ICAM-1-Biotin,Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at aconcentration, of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody).Cells are incubated at 37° C. for 30 min. in a humidified environment.Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. 20 μl of dilutedExtrAvidin-Alkaline Phosphatase (1:5,000 dilution, referred to herein asthe working dilution) are added to each well and incubated at 37° C. for30 min. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. Dissolve1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH10.4). 100 μl of pNPP substrate in glycine buffer is added to each testwell. Standard wells in triplicate are prepared from the workingdilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer:1:5,000 (10⁰)>10^(−0.5)>10⁻¹>10^(−1.5). 5 μl of each dilution is addedto triplicate wells and the resulting AP content in each well is 5.50ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added toeach of the standard wells. The plate is incubated at 37° C. for 4 h. Avolume of 50 μl of 3M NaOH is added to all wells. The plate is read on aplate reader at 405 nm using the background subtraction option on blankwells filled with glycine buffer only. Additionally, the template is setup to indicate the concentration of AP-conjugate in each standard well[5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount ofbound AP-conjugate in each sample.

Example 41 Alamar Blue Endothelial Cells Proliferation Assay

This assay may be used to quantitatively determine protein mediatedinhibition of bFGF-induced proliferation of Bovine Lymphatic EndothelialCells (LECs), Bovine Aortic Endothelial Cells (BAECs) or HumanMicrovascular Uterine Myometrial Cells (UTMECs). This assay incorporatesa fluorometric growth indicator based on detection of metabolicactivity. A standard Alamar Blue Proliferation Assay is prepared inEGM-2MV with 10 ng/ml of bFGF added as a source of endothelial cellstimulation. This assay may be used with a variety of endothelial cellswith slight changes in growth medium and cell concentration. Dilutionsof protein batches to be tested are diluted as appropriate. Serum-freemedium (GIBCO SFM) without bFGF is used as a non-stimulated control andAngiostatin or TSP-1 are included as a known inhibitory controls.

Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of5000 to 2000 cells/well in a 96 well plate and placed at 37 degrees Covernight. After the overnight incubation of the cells, the growth mediais removed and replaced with GIBCO EC-SFM. The cells are treated withthe appropriate dilutions of an albumin. fusion protein of the inventionor control protein sample(s) (prepared in SFM) in triplicate wells withadditional bFGF to a concentration of 10 ng/ml. Once the cells have beentreated with the samples, the plate(s) is/are placed back in the 37° C.incubator for three days. After three days 10 ml of stock alamar blue(Biosource Cat# DAL1100) is added to each well and the plate(s) is/areplaced back in the 37° C. incubator for four hours. The plate(s) arethen read at 530 nm excitation and 590 nm emission using the CytoFluorfluorescence reader. Direct output is recorded in relative fluorescenceunits.

Alamar blue is an oxidation-reduction indicator that both fluoresces andchanges color in response to chemical reduction of growth mediumresulting from cell growth. As cells grow in culture, innate metabolicactivity results in a chemical reduction of the immediate surroundingenvironment. Reduction related to growth causes the indicator to changefrom oxidized (non-fluorescent blue) form to reduced (fluorescent red)form (i.e., stimulated proliferation will produce a stronger signal andinhibited proliferation will produce a weaker signal and the totalsignal is proportional to the total number of cells as well as theirmetabolic activity). The background level of activity is observed withthe starvation medium alone. This is compared to the output observedfrom the positive control samples (bFGF in growth medium) and proteindilutions.

Example 42 Detection of Inhibition of a Mired Lymphocyte Reaction

This assay can be used to detect and evaluate inhibition of a MixedLymphocyte Reaction (MLR) by fusion proteins of the invention.Inhibition of a MLR may be due to a direct effect on cell proliferationand viability, modulation of costimulatory molecules on interactingcells, modulation of adhesiveness between lymphocytes and accessorycells, or modulation of cytokine production by accessory cells. Multiplecells may be targeted by the albumin fusion proteins that inhibit MLRsince the peripheral blood mononuclear fraction used in this assayincludes T, B and natural killer lymphocytes, as well as monocytes anddendritic cells.

Albumin fusion proteins of the invention found to inhibit the MLR mayfind application in diseases associated with lymphocyte and monocyteactivation or proliferation. These include, but are not limited to,diseases such as asthma, arthritis, diabetes, inflammatory skinconditions, psoriasis, eczema, systemic lupus erythematosus, multiplesclerosis, glomerulonephritis, inflammatory bowel disease, crohn'sdisease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. hostdisease, host vs. graft disease, hepatitis, leukemia and lymphoma.

Briefly, PBMCs from human donors are purified by density gradientcentrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from twodonors are adjusted to 2×10⁶ cells/ml in RPMI-1640 (Life Technologies,Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCsfrom a third donor is adjusted to 2×10⁵ cells/ml. Fifty microliters ofPBMCs from each donor is added to wells of a 96-well round bottommicrotiter plate. Dilutions of the fusion protein test material (50 μl)is added in triplicate to microtiter wells. Test samples (of the proteinof interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems,Minneapolis, Minn., catalog number 202-IL) is added to a finalconcentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11,catalog number MAB379) is added to a final concentration of 10 μg/ml.Cells are cultured for 7-8 days at 37° C. in 5% CO₂, and 1 μC of [³H]thymidine is added to wells for the last 16 hrs of culture. Cells areharvested and thymidine incorporation determined using a PackardTopCount. Data is expressed as the mean and standard deviation oftriplicate determinations.

Samples of the fusion protein of interest are screened in separateexperiments and compared to the negative control treatment, anti-CD4mAb, which inhibits proliferation of lymphocytes and the positivecontrol treatment, IL-2 (either as recombinant material or supernatant),which enhances proliferation of lymphocytes.

Example 43 Assays for Protease Activity

The following assay may be used to assess protease activity of analbumin fusion protein of the invention.

Gelatin and casein zymoaraphy are performed essentially as described(Heusen et al., Anal. Biochem., 102:196-202 (1980); Wilson et al.,Journal of Urology, 149:653-658 (1993)). Samples are run on 10%polyacryamide/0.1% SDS gels containing 1% gelain orcasein, soaked in2.5% triton at room temperature for 1 hour, and in 0.1 M glycine, pH 8.3at 37° C. 5 to 16 hours. After staining in amido black areas ofproteolysis apear as clear areas agains the blue-black background.Trypsin (Sigma T8642) is used as a positive control.

Protease activity is also determined by monitoring the cleavage ofn-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B4500. Reactions areset up in (25 mMNaPO₄, 1 mM EDTA, and 1 mM BAEE), pH 7.5. Samples areadded and the change in adsorbance at 260 nm is monitored on the BeckmanDU-6 spectrophotometer in the time-drive mode. Trypsin is used as apositive control.

Additional assays based upon the release of acid-soluble peptides fromcasein or hemoglobin measured as adsorbance at 280 nm orcolorimetrically using the Folin method are performed as described inBergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984). Other assaysinvolve the solubilization of chromogenic substrates (Ward, AppliedScience, 251-317 (1983)).

Example 44 Identifying Serine Protease Substrate Specificity

Methods known in the art or described herein may be used to determinethe substrate specificity of the albumin fusion proteins of the presentinvention having serine protease activity. A preferred method ofdetermining substrate specificity is by the use of positional scanningsynthetic combinatorial libraries as described in GB 2 324 529(incorporated herein in its entirety).

Example 45 Ligand Binding Assays

The following assay may be used to assess ligand binding activity of analbumin fusion protein of the invention.

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. The purifiedligand for an albumin fusion protein of the invention is radiolabeled tohigh specific activity (50-2000 Ci/mmol) for binding studies. Adetermination is then made that the process of radiolabeling does notdiminish the activity of the ligand towards the fusion protein. Assayconditions for buffers, ions, pH and other modulators such asnucleotides are optimized to establish a workable signal to noise ratiofor both membrane and whole cell polypeptide sources. For these assays,specific polypeptide binding is defined as total associatedradioactivity minus the radioactivity measured in the presence of anexcess of unlabeled competing ligand. Where possible, more than onecompeting ligand is used to define residual nonspecific binding.

Example 46 Functional Assay in Xenopus Oocytes

Capped RNA transcripts from linearized plasmid templates encoding analbumin fusion protein of the invention is synthesized in vitro with RNApolymerases in accordance with standard procedures. In vitro transcriptsare suspended in water at a final concentration of 0.2 mg/ml. Ovarianlobes are removed from adult female toads, Stage V defolliculatedoocytes are obtained, and RNA transcripts (10 ng/oocytc) are injected ina 50 nl bolus using a microinjection apparatus. Two electrode voltageclamps are used to measure the currents from individual Xenopus oocytesin response fusion protein and polypeptide agonist exposure. Recordingsare made in Ca2+ free Barth's medium at room temperature. The Xenopussystem can be used to screen known ligands and tissue/cell extracts foractivating ligands.

Example 47 Microphysiometric Assays

Activation of a wide variety of secondary messenger systems results inextrusion of small amounts of acid from a cell. The acid formed islargely as a result of the increased metabolic activity required to fuelthe intracellular signaling process. The pH changes in the mediasurrounding the cell are very small but are detectable by the CYTOSENSORmicrophysiometer (Molecular Devices Ltd., Menlo Park, Calif.). TheCYTOSENSOR is thus capable of detecting the ability of an albumin fusionprotein of the invention to activate secondary messengers that arecoupled to an energy utilizing intracellular signaling pathway.

Example 48 Extract/Cell Supernatant Screening

A large number of mammalian receptors exist for which there remains, asyet, no cognate activating ligand (agonist). Thus, active ligands forthese receptors may not be included within the ligands banks asidentified to date. Accordingly, the albumin fusion proteins of theinvention can also be functionally screened (using calcium, cAMP,microphysiometer, oocyte electrophysiology, etc., functional screens)against tissue extracts to identify natural ligands for the Therapeuticprotein portion and/or albumin protein portion of an albumin fusionprotein of the invention. Extracts that produce positive functionalresponses can be sequentially subfractionated until an activating ligandis isolated and identified.

Example 49 ATP-Binding Assay

The following assay may be used to assess ATP-binding activity of fusionproteins of the invention.

ATP-binding activity of an albumin fusion protein of the invention maybe detected using the ATP-binding assay described in U.S. Pat. No.5,853,719, which is herein incorporated by reference in its entirety.Briefly, ATP-binding to an albumin fusion protein of the invention ismeasured via photoaffinity labeling with 8-azido-ATP in a competitionassay. Reaction mixtures containing 1 mg/ml of ABC transport protein areincubated with varying concentrations of ATP, or the non-hydrolyzableATP analog adenyl-5′-imidodiphosphate for 10 minutes at 4° C. A mixtureof 8-azido-ATP (Sigma Chem. Corp., St. Louis, Mo.) plus 8-azido-ATP(³²P-ATP) (5 mCi/μmol, ICN, Irvine Calif.) is added to a finalconcentration of 100 μM and 0.5 ml aliquots are placed in the wells of aporcelain spot plate on ice. The plate is irradiated using a short wave254 nm UV lamp at a distance of 2.5 cm from the plate for two one-minuteintervals with a one-minute cooling interval in between. The reaction isstopped by addition of dithiothreitol to a final concentration of 2 mM.The incubations are subjected to SDS-PAGE electrophoresis, dried, andautoradiographed. Protein bands corresponding to the albumin fusionproteins of the invention are excised, and the radioactivity quantified.A decrease in radioactivity with increasing ATP oradenly-5′-imidodiphosphate provides a measure of ATP affinity to thefusion protein.

Example 50 Phosphorylation Assay

In order to assay for phosphorylation activity of an albumin fusionprotein of the invention, a phosphorylation assay as described in U.S.Pat. No. 5,958,405 (which is herein incorporated by reference) isutilized. Briefly, phosphorylation activity may be measured byphosphorylation of a protein substrate using gamma-labeled ³²P-ATP andquantitation of the incorporated radioactivity using a gammaradioisotope counter. The fusion portein of the invention is incubatedwith the protein substrate, ³²P-ATP, and a kinase buffer. The ³²Pincorporated into the substrate is then separated from free ³²P-ATP byelectrophoresis, and the incorporated ³²P is counted and compared to anegative control. Radioactivity counts above the negative control areindicative of phosphorylation activity of the fusion protein.

Example 51 Detection of Phosphorylation Activity (Activation) of anAlbumin Fusion Protein of the Invention in the Presence of PolypeptideLigands

Methods known in the art or described herein may be used to determinethe phosphorylation activity of an albumin fusion protein of theinvention. A preferred method of determining phosphorylation activity isby the use of the tyrosine phosphorylation assay as described in U.S.Pat. No. 5,817,471 (incorporated herein by reference).

Example 52 Identification of Signal Transduction Proteins that Interactwith an Albumin Fusion Protein of the Present Invention

Albumin fusion proteins of the invention may serve as research tools forthe identification, characterization and purification of signaltransduction pathway proteins or receptor proteins. Briefly, a labeledfusion protein of the invention is useful as a reagent for thepurification of molecules with which it interacts. In one embodiment ofaffinity purification, an albumin fusion protein of the invention iscovalently coupled to a chromatography column. Cell-free extract derivedfrom putative target cells, such as carcinoma tissues, is passed overthe column, and molecules with appropriate affinity bind to the albuminfusion protein. The protein complex is recovered from the column,dissociated, and the recovered molecule subjected to N-terminal proteinsequencing. This amino acid sequence is then used to identify thecaptured molecule or to design degenerate oligonucleotide probes forcloning the relevant gene from an appropriate cDNA library.

Example 53 IL-6 Bioassay

A variety of assays are known in the art for testing the proliferativeeffects of an albumin fusion protein of the invention. For example, onesuch asssay is the IL-6 Bioassay as described by Marz et al. (Proc.Natl. Acad. Sci., U.S.A., 95:3251-56 (1998), which is hereinincorporated by reference). After 68 hrs. at 37° C., the number ofviable cells is measured by adding the tetrazolium salt thiazolyl blue(MTT) and incubating for a further 4 hrs. at 37° C. B9 cells are lysedby SDS and optical density is measured at 570 nm. Controls containingIL-6 (positive) and no cytokine (negative) are Briefly, IL-6 dependentB9 murine cells are washed three times in IL-6 free medium and plated ata concentration of 5,000 cells per well in 50 μl, and 50 μl of fusionprotein of the invention is added utilized. Enhanced proliferation inthe test sample(s) (containing an albumin fusion protein of theinvention) relative to the negative control is indicative ofproliferative effects mediated by the fusion protein.

Example 54 Support of Chicken Embryo Neuron Survival

To test whether sympathetic neuronal cell viability is supported by analbumin fusion protein of the invention, the chicken embryo neuronalsurvival assay of Senaldi et al may be utilized (Proc. Natl. Acad. Sci.,U.S.A., 96:11458-63 (1998), which is herein incorporated by reference).Briefly, motor and sympathetic neurons are isolated from chickenembryos, resuspended in L15 medium (with 10% FCS, glucose, sodiumselenite, progesterone, conalbumin, putrescine, and insulin; LifeTechnologies, Rockville, Md.) and Dulbecco's modified Eagles medium[with 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH 7.2);Life Technologies, Rockville, Md.], respectively, and incubated at 37°C. in 5% CO₂ in the presence of different concentrations of the purifiedfusion protein of the invention, as well as a negative control lackingany cytokine. After 3 days, neuron survival is determined by evaluationof cellular morphology, and through the use of the colorimetric assay ofMosmann (Mosmann, T., J. Immunol. Methods, 65:55-63 (1983)). Enhancedneuronal cell viability as compared to the controls lacking cytokine isindicative of the ability of the albumin fusion protein to enhance thesurvival of neuronal cells.

Example 55 Assay for Phosphatase Activity

The following assay may be used to assess serine/threonine phosphatase(PTPase) activity of an albumin fusion protein of the invention.

In order to assay for serine/threonine phosphatase (PTPase) activity,assays can be utilized which are widely known to those skilled in theart. For example, the serine/threonine phosphatase (PSPase) activity ofan albumin fusion protein of the invention may be measured using aPSPase assay kit from New England Biolabs, Inc. Myelin basic protein(MyBP), a substrate for PSPase, is phosphorylated on serine andthreonine residues with cAMP-dependent Protein Kinase in the presence of[³²P]ATP. Protein serine/threonine phosphatase activity is thendetermined by measuring the release of inorganic phosphate from³²P-labeled MyBP.

Example 56 Interaction of Serine/Threonine Phosphatases with OtherProteins

Fusion protein of the invention having serine/threonine phosphataseactivity (e.g., as determined in Example 55) are useful, for example, asresearch tools for the identification, characterization and purificationof additional interacting proteins or receptor proteins, or other signaltransduction pathway proteins. Briefly, a labeled fusion protein of theinvention is useful as a reagent for the purification of molecules withwhich it interacts. In one embodiment of affinity purification, analbumin fusion protein of the invention is covalently coupled to achromatography column. Cell-free extract derived from putative targetcells, such as neural or liver cells, is passed over the column, andmolecules with appropriate affinity bind to the fusion protein. Thefusion protein complex is recovered from the column, dissociated, andthe recovered molecule subjected to N-terminal protein sequencing. Thisamino acid sequence is then used to identify the captured molecule or todesign degenerate oligonucleotide probes for cloning the relevant genefrom an appropriate cDNA library.

Example 57 Assaying for Heparanase Activity

There a numerous assays known in the art that may be employed to assayfor heparanase activity of an albumin fusion protein of the invention.In one example, heparanase activity of an albumin fusion protein of theinvention, is assayed as described by Vlodavsky et al., (Vlodavsky etal., Nat. Med., 5:793-802 (1999)). Briefly, cell lysates, conditionedmedia, intact cells (1×10⁶ cells-per 35-mm dish), cell culturesupernatant, or purified fusion protein are incubated for 18 hrs at 37°C., pH 6.2-6.6, with ³⁵S-labeled ECM or soluble ECM derived peak Iproteoglycans. The incubation medium is centrifuged and the supernatantis analyzed by gel filtration on a Sepharose CL-6B column (0.9×30 cm).Fractions are eluted with PBS and their radioactivity is measured.Degradation fragments of heparan sulfate side chains are eluted fromSepharose 6B at 0.5<K_(av), <0.8 (peak II). Each experiment is done atleast three times. Degradation fragments corresponding to “peak II,” asdescribed by Vlodavsky et al., is indicative of the activity of analbumin fusion protein of the invention in cleaving heparan sulfate.

Example 58 Immobilization of Biomolecules

This example provides a method for the stabilization of an albuminfusion protein of the invention in non-host cell lipid bilayer constucts(see, e.g., Bieri et al., Nature Biotech 17:1105-1108 (1999), herebyincorporated by reference in its entirety herein) which can be adaptedfor the study of fusion proteins of the invention in the variousfunctional assays described above. Briefly, carbohydrate-specificchemistry for biotinylation is used to confine a biotin tag to analbumin fusion protein of the invention, thus allowing uniformorientation upon immobilization. A 50 uM solution of an albumin fusionprotein of the invention in washed membranes is incubated with 20 mMNaIO4 and 1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5mM) biotin-hydrazide for1 hr at room temperature (reaction volume, 150 ul). Then the sample isdialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce ChemicalCo., Rockford Ill.) at 4 C first for 5 h, exchanging the buffer aftereach hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCl, 1mM MgCl2, 10 mM sodium phosphate, pH7). Just before addition into acuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer Rsupplemented with 50 mM octylglucoside).

Example 59 Assays for Metalloproteinase Activity

Metalloproteinases are peptide hydrolases which use metal ions, such asZn²⁺, as the catalytic mechanism. Metalloproteinase activity of analbumin fusion protein of the present invention can be assayed accordingto methods known in the art. The following exemplary methods areprovided:

Proteolysis of Alpha-2-Macroglobulin

To confirm protease activity, a purified fusion protein of the inventionis mixed with the substrate alpha-2-macroglobulin (0.2 unit/ml;Boehringer Mannheim, Germany) in 1× assay buffer (50 mM HEPES, pH 7.5,0.2 M NaCl, 10 mM CaCl₂, 25 μM ZnCl₂ and 0.05% Brij-35) and incubated at37° C. for 1-5 days. Trypsin is used as positive control. Negativecontrols contain only alpha-2-macroglobulin in assay buffer. The samplesare collected and boiled in SDS-PAGE sample buffer containing 5%2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide gel.After electrophoresis the proteins are visualized by silver staining.Proteolysis is evident by the appearance of lower molecular weight bandsas compared to the negative control.

Inhibition of Alpha-2-Macroglobulin Proteolysis by Inhibitors ofMetalloproteinases

Known metalloproteinase inhibitors (metal chelators (EGTA, EGTA, ANDHgCl₂), peptide metalloproteinase inhibitors (TIMP-1 and TIMP-2), andcommercial small molecule MMP inhibitors) may also be used tocharacterize the proteolytic activity of an albumin fusion protein ofthe invention. Three synthetic MMP inhibitors that may be used are: MMPinhibitor I, [IC₅₀=1.0 μM against MMP-8 and MMP-8; IC₅₀=30 μM againstMMP-9; IC₅₀=150 μM against MMP-3]; MMP-3 (stromelysin-1) inhibitor I[IC₅₀=5 μM against MMP-3], and MMP-3 inhibitor II [K_(i)=130 nM againstMMP-3]; inhibitors available through Calbiochem, catalog # 444250,444218, and 444225, respectively). Briefly, different concentrations ofthe small molecule MMP inhibitors are mixed with a purified fusionprotein of the invention (50 μg/ml) in 22.9 μl of 1× HEPES buffer (50 mMHEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl₂, 25 μM ZnCl₂ and 0.05% Brij-35)and incubated at room temperature (24° C.) for 2-hr, then 7.1 μl ofsubstrate alpha-2-macroglobulin (0.2 unit/ml) is added and incubated at37° C. for 20-hr. The reactions are stopped by adding 4× sample bufferand boiled immediately for 5 minutes. After SDS-PAGE, the protein bandsare visualized by silver stain.

Synthetic Fluorogenic Peptide Substrates Cleavage Assay

The substrate specificity for fusion proteins of the invention withdemonstrated metalloproteinase activity may be determined usingtechniques knonw in the art, such as using synthetic fluorogenic peptidesubstrates (purchased from BACHEM Bioscience Inc). Test substratesinclude, M-1985, M-2225, M-2105, M-2110, and M-2255. The first four areMMP substrates and the last one is a substrate of tumor necrosisfactor-α (TNF-α) converting enzyme (TACE). These substrastes arepreferably prepared in 1:1 dimethyl sulfoxide (DMSO) and water. Thestock solutions are 50-500 4M. Fluorescent assays are performed by usinga Perkin Elmer LS 50B luminescence spectrometer equipped with a constanttemperature water bath. The excitation λ is 328 nm and the emission λ is393 nm. Briefly, the assay is carried out by incubating 176 μl 1× HEPESbuffer (0.2 M NaCl, 10 mMi CaCl₂, 0.05% Brij-35 and 50 mM HEPES, pH 7.5)with 4 μl of substrate solution (50 μM) at 25° C. for 15 minutes, andthen adding 20 μl of a purified fusion protein of the invention into theassay cuvett. The final concentration of substrate is 1 μM. Initialhydrolysis rates are monitored for 30-min.

Example 60 Identification and Cloning of VH and VL Domains

One method to identfy and clone VH and VL domains from cell linesexpressing a particular antibody is to perform PCR with VH and VLspecific primers on cDNA made from the antibody expressing cell lines.Briefly, RNA is isolated from the cell lines and used as a template forRT-PCR designed to amplify the VH and VL domains of the antibodiesexpressed by the EBV cell lines. Cells may be lysed in the TRIzol®reagent (Life Technologies, Rockville. MD) and extracted with one fifthvolume of chloroform. After addition of chloroform, the solution isallowed to incubate at room temperature for 10 minutes, and thecentrifuged at 14,000 rpm for 15 minutes at 4° C. in a tabletopcentrifuge. The supernatant is collected and RNA is precipitated usingan equal volume of isopropanol. Precipitated RNA is pelleted bycentrifuging at 14,000 rpm for 15 minutes at 4° C. in a tabletopcentrifuge. Following centrifugation, the supernatant is discarded andwashed with 75% ethanol. Follwing washing, the RNA is centrifuged againat 800 rpm for 5 minutes at 4° C. The supernatant is discarded and thepellet allowed to air dry. RNA is the dissolved in DEPC water and heatedto 60° C. for 10 minutes. Quantities of RNA can determined using opticaldensity measurements.

cDNA may be synthesized, according to methods well-known in the art,from 1.5-2.5 micrograms of RNA using reverse transciptase and randomhexamer primers. cDNA is then used as a template for PCR amplificationof VH and VL domains. Primers used to amplify VH and VL genes are shownin Table 3. Typically a PCR reaction makes use of a single 5′ primer anda single 3′ primer. Sometimes, when the amount of available RNA templateis limiting, or for greater efficiency, groups of 5′ and/or 3′ primersmay be used. For example, sometimes all five VH-5′ primers and all JH3′primers are used in a single PCR reaction. The PCR reaction is carriedout in a 50 microliter volume containing 1× PCR buffer, 2 mM of eachdNTP, 0.7 units of High Fidelity Taq polymerse, 5′ primer mix, 3′ primermix and 7.5 microliters of cDNA. The 5′ and 3′ primer mix of both VH andVL can be made by pooling together 22 pmole and 23 pmole, respectively,of each of the individual primers. PCR conditions are: 96° C. for 5minutes; followed by 25 cycles of 94° C. for 1 minute, 50° C. for 1minute, and 72° C. for 1 minute; followed by an extension cycle of 72°C. for 10 minutes. After the reaction is completed, sample tubes arestored 4° C. TABLE 3 Primer Sequences Used to Amplify VH and VL domains.SEQ ID Primer name NO Primer Sequence (5′-3′) VH Primers Hu VH1-5′ 36CAGGTGCAGCTGGTGCAGTCTGG Hu VH2-5′ 37 CAGGTCAACTTAAGGGAGTCTGG Hu VH3-5′38 GAGGTGCAGCTGGTGGAGTCTGG Hu VH4-5′ 39 CAGGTGCAGCTGCAGGAGTCGGG HuVH5-5′ 40 GAGGTGCAGCTGTTGCAGTCTGC Hu VH6-5′ 41 CAGGTACAGCTGCAGCAGTCAGGHu JH1,2-5′ 42 TGAGGAGACGGTGACCAGGGTGCC Hu JH3-5′ 43TGAAGAGACGGTGACCATTGTCCC Hu JH4,5-5′ 44 TGAGGAGACGGTGACCAGGGTTCC HuJH6-5′ 45 TGAGGAGACGGTGACCGTGGTCCC VL Primers Hu Vkappa1-5′ 46GACATCCAGATGACCCAGTCTCC Hu Vkappa2a-5′ 47 GATGTTGTGATGACTCAGTCTCC HuVkappa2b-5′ 48 GATATTGTGATGACTCAGTCTCC Hu Vkappa3-5′ 49GAAATTGTGTTGACGCAGTCTCC Hu Vkappa4-5′ 50 GACATCGTGATGACCCAGTCTCC HuVkappa5-5′ 51 GAAACGACACTCACGCAGTCTCC Hu Vkappa6-5′ 52GAAATTGTGCTGACTCAGTCTCC Hu Vlambda1-5′ 53 CAGTCTGTGTTGACGCAGCCGCC HuVlambda2-5′ 54 CAGTCFGCCCTGACTCAGCCTGC Hu Vlambda3-5′ 55TCCTATGTGCTGACTCAGCCACC Hu Vlambda3b-5′ 56 TCTTCTGAGCTGACTCAGGACCC HuVlambda4-5′ 57 CACGTTATACTGACTCAACCGCC Hu Vlambda5-5′ 58CAGGCTGTGCTCACTCAGCCGTC Hu Vlambda6-5′ 59 AATTTTATGCTGACTCAGCCCCA HuJkappa1-3′ 60 ACGTTTGATTTCCACCTTGGTCCC Hu Jkappa2-3′ 61ACGTTTGATCTCCAGCTTGGTCCC Hu Jkappa3-3′ 62 ACGTTTGATATCCACTTTGGTCCC HuJkappa4-3′ 63 ACGTTTGATCTCCACCTTGGTCCC Hu Jkappa5-3′ 64ACGTTTAATCTCCAGTCGTGTCCC Hu Jlambda1-3′ 65 CAGTCTGTGTTGACGCAGCCGCC HuJlambda2-3′ 66 CAGTCTGCCCTGACTCAGCCTGC Hu Jlambda3--3′ 67TCCTATGTGCTGACTCAGCCACC Hu Jlambda3b-3′ 68 TCTTCTGAGCTGACTCAGGACCC HuJlambda4-3′ 69 CACGTTATACTGACTCAACCGCC Hu Jlambda5-3′ 70CAGGCTGTGCTCACTCAGCCGTC Hu Jlambda6-3′ 71 AATTTTATGCTGACTCAGCCCCA

PCR samples are then electrophoresed on a 1.3% o agarose gel. DNA bandsof the expected sizes (˜506 base pairs for VH domains, and 344 basepairs for VL domains) can be cut out of the gel and purified usingmethods well known in the art. Purified PCR products can be ligated intoa PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad, Calif.).Individual cloned PCR products can be isolated after transfection of E.coli and blue/white color selection. Cloned PCR products may then besequenced using methods commonly known in the art.

The PCR bands containing the VH domain and the VL domains can also beused to create full-length 1 g expression vectors. VH and VL domains canbe cloned into vectors containing the nucleotide sequences of a heavy(e.g., human IcG1 or human IgG4) or light chain (human kappa or humanlambda) constant regions such that a complete heavy or light chainmolecule could be expressed from these vectors when transfected into anappropriate host cell. Further, when cloned heavy and light chains areboth expressed in one cell line (from either one or two vectors), theycan assemble into a complete functional antibody molecule that issecreted into the cell culture medium. Methods using polynucleotidesencoding VH and VL antibody domain to generate expression vectors thatencode complete antibody molecules are well known within the art.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, patent publications, journal articles, abstracts,laboratory manuals, books, or other disclosures) as well as informationavailable through Identifiers specific to databases such as GenBank,GeneSeq, or the CAS Registry, referred to in this application are hereinincorporated by reference in their entirety. The specification andsequence listing of each of the following U.S. applications are hereinincorporated by reference in their entirety: application Ser. Nos.09/091,8773 filed Jun. 25, 1998; 60/229,358 filed on Apr. 12, 2000;60/199,384 filed on Apr. 25, 2000 and 60/256,931 filed on Dec. 21, 2000.LENGTHY TABLE The patent application contains a lengthy table section. Acopy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070026013A1)An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. An albumin fusion protein comprising a member selected from: (a) aTherapeutic protein X and albumin comprising the amino acid sequence ofSEQ ID NO:18; (b) a Therapeutic protein X and a fragment or a variant ofthe amino acid sequence of SEQ ID NO:18, wherein said fragment orvariant has albumin activity; (c) a Therapeutic protein X and a fragmentor a variant of the amino acid sequence of SEQ ID NO:18, wherein saidfragment or variant has albumin activity, and further wherein saidalbumin activity is the ability to prolong the shelf life of theTherapeutic protein X compared to the shelf-life of the Therapeuticprotein X in an unfused state; (d) a Therapeutic protein X and afragment or a variant of the amino acid sequence of SEQ ID NO:18,wherein said fragment or variant has albumin activity, and furtherwherein said albumin activity is the ability to increase the half-lifeof the Therapeutic protein X compared to the half-life of theTherapeutic protein X in an unfused state; (e) a Therapeutic protein Xand a fragment or a variant of the amino acid sequence of SEQ ID NO:18,wherein said fragment or variant has albumin activity, and furtherwherein the fragment or variant comprises the amino acid sequence ofamino acids 1-387 of SEQ ID NO:18; (f) a fragment or variant of aTherapeutic protein X and albumin comprising the amino acid sequence ofSEQ ID NO:18, wherein said fragment or variant has a biological activityof the Therapeutic protein X; (g) a Therapeutic protein X, or fragmentor variant thereof, and albumin, or fragment or variant thereof, of (a)to (f), wherein the Therapeutic protein X, or fragment or variantthereof, is fused to the N-terminus of albumin, or the N-terminus of thefragment or variant of albumin; (h) a Therapeutic protein X, or fragmentor variant thereof, and albumin, or fragment or variant thereof, of (a)to (f), wherein the Therapeutic protein X, or fragment or variantthereof, is fused to the C-terminus of albumin, or the C-terminus of thefragment or variant of albumin; (i) a Therapeutic protein X, or fragmentor variant thereof, and albumin, or fragment or variant thereof, of (a)to (f), wherein the Therapeutic protein X, or fragment or variantthereof, is fused to the N-terminus and C-terminus of albumin, or theN-terminus and the C-terminus of the fragment or variant of albumin; (j)a Therapeutic protein X, or fragment or variant thereof, and albumin, orfragment or variant thereof, of (a) to (f), which comprises a firstTherapeutic protein X, or fragment or variant thereof, and a secondTherapeutic protein X, or fragment or variant thereof, wherein saidfirst Therapeutic protein X, or fragment or variant thereof, isdifferent from said second Therapeutic protein X, or fragment or variantthereof; (k) a Therapeutic protein X, or fragment or variant thereof,and albumin, or fragment or variant thereof, of (a) to (j), wherein theTherapeutic protein X, or fragment or variant thereof, is separated fromthe albumin or the fragment or variant of albumin by a linker; and (l) aTherapeutic protein X, or fragment or variant thereof, and albumin, orfragment or variant thereof, of (a) to (k), wherein the albumin fusionprotein has the following formula:R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, and further wherein R1 is TherapeuticProtein X, or fragment or variant thereof, L is a peptide linker, and R2is albumin comprising the amino acid sequence of SEQ ID NO:18 or afragment or variant of albumin.
 2. The albumin fusion protein of claim1, wherein the shelf-life of the albumin fusion protein is greater thanthe shelf-life of the Therapeutic protein X, or fragment or variantthereof, in an unfused state.
 3. The albumin fusion protein of claim 1,wherein the in vitro biological activity of the Therapeutic protein X,or fragment or variant thereof, fused to albumin, or fragment or variantthereof, is greater than the in vitro biological activity of theTherapeutic protein X, or fragment or variant thereof, in an unfusedstate.
 4. The albumin fusion protein of claim 1, wherein the in vivobiological activity of the Therapeutic protein X, or fragment or variantthereof, fused to albumin, or fragment or variant thereof, is greaterthan the in vivo biological activity of the Therapeutic protein X, orfragment or variant thereof, in an unfused state.
 5. An albumin fusionprotein comprising a Therapeutic protein X, or fragment or variantthereof, inserted into an albumin, or fragment or variant thereof,comprising the amino acid sequence of SEQ ID NO:18 or fragment orvariant thereof.
 6. An albumin fusion protein comprising a Therapeuticprotein X, or fragment or variant thereof, inserted into an albumin, orfragment or variant thereof, comprising an amino acid sequence selectedfrom: (a) amino acids 54 to 61 of SEQ ID NO:18; (b) amino acids 76 to 89of SEQ ID NO:18; (c) amino acids 92 to 100 of SEQ ID NO:18; (d) aminoacids 170 to 176 of SEQ ID NO:18; (e) amino acids 247 to 252 of SEQ IDNO:18; (f) amino acids 266 to 277 of SEQ ID NO:18; (g) amino acids 280to 288 of SEQ ID NO:18; (h) amino acids 362 to 368 of SEQ ID NO:18; (i)amino acids 439 to 447 of SEQ ID NO:18; (j) amino acids 462 to 475 ofSEQ ID NO:18; (k) amino acids 478 to 486 of SEQ ID NO:18; and (l) aminoacids 560 to 566 of SEQ ID NO:18.
 7. The albumin fusion protein of claim5, wherein said albumin fusion protein comprises a portion of albuminsufficient to prolong the shelf-life of the Therapeutic protein X, orfragment or variant thereof, as compared to the shelf-life of theTherapeutic protein X, or fragment or variant thereof, in an unfusedstate.
 8. The albumin fusion protein of claim 6, wherein said albuminfusion protein comprises a portion of albumin sufficient to prolong theshelf-life of the Therapeutic protein X, or fragment or variant thereof,as compared to the shelf-life of the Therapeutic protein X, or fragmentor variant thereof, in an unfused state.
 9. The albumin fusion proteinof claim 5, wherein said albumin fusion protein comprises a portion ofalbumin sufficient to prolong the in vitro biological activity of theTherapeutic protein X, or fragment or variant thereof, fused to albuminas compared to the in vitro biological activity of the Therapeuticprotein X, or fragment or variant thereof, in an unfused state.
 10. Thealbumin fusion protein of claim 6, wherein said albumin fusion proteincomprises a portion of albumin sufficient to prolong the in vitrobiological activity of the Therapeutic protein X, or fragment or variantthereof, fused to albumin as compared to the in vitro biologicalactivity of the Therapeutic protein X, or fragment or variant thereof,in an unfused state.
 11. The albumin fusion protein of claim 5 whereinsaid albumin fusion protein comprises a portion of albumin sufficient toprolong the in vivo biological activity of the Therapeutic protein X, orfragment or variant thereof, fused to albumin compared to the in vivobiological activity of the Therapeutic protein X, or fragment or variantthereof, in an unfused state.
 12. The albumin fusion protein of claim 6wherein said albumin fusion protein comprises a portion of albuminsufficient to prolong the in vivo biological activity of the Therapeuticprotein X, or fragment or variant thereof, fused to albumin compared tothe in vivo biological activity of the Therapeutic protein X, orfragment or variant thereof, in an unfused state.
 13. The albumin fusionprotein of claim 1, which is non-glycosylated.
 14. The albumin fusionprotein of claim 1, which is expressed in yeast.
 15. The albumin fusionprotein of claim 14, wherein the yeast is glycosylation deficient. 16.The albumin fusion protein of claim 14 wherein the yeast isglycosylation and protease deficient.
 17. The albumin fusion protein ofclaim 1, which is expressed by a mammalian cell.
 18. The albumin fusionprotein of claim 1, wherein the albumin fusion protein is expressed by amammalian cell in culture.
 19. The albumin fusion protein of claim 1,wherein the albumin fusion protein further comprises a secretion leadersequence.
 20. A composition comprising the albumin fusion protein ofclaim 1 and a pharmaceutically acceptable carrier.
 21. A kit comprisingthe composition of claim
 20. 22. A method of treating a disease ordisorder in a patient, comprising the step of administering the albuminfusion protein of claim
 1. 23. The method of claim 22, wherein thedisease or disorder comprises indication Y.
 24. A method of treating apatient with a disease or disorder that is modulated by Therapeuticprotein X, or fragment or variant thereof, comprising the step ofadministering an effective amount of the albumin fusion protein ofclaim
 1. 25. The method of claim 24, wherein the disease or disorder isindication Y.
 26. A method of extending the shelf life of Therapeuticprotein X, or fragment or variant thereof, comprising the step of fusingthe Therapeutic protein X, or fragment or variant thereof, to albumin,or fragment or variant thereof, sufficient to extend the shelf-life ofthe Therapeutic protein X, or fragment or variant thereof, compared tothe shelf-life of the Therapeutic protein X, or fragment or variantthereof, in an unfused state.
 27. A nucleic acid molecule comprising apolynucleotide sequence encoding the albumin fusion protein of claim 1.28. A vector comprising the nucleic acid molecule of claim
 27. 29. Ahost cell comprising the nucleic acid molecule of claim
 28. 30. Thealbumin fusion protein of claim 1, wherein the half-life of the albuminfusion protein is greater than the half-life of the Therapeutic proteinX, or fragment or variant thereof, in an unfused state.
 31. The albuminfusion protein of claim 5, wherein said albumin fusion protein comprisesa portion of albumin sufficient to increase the half-life of theTherapeutic protein X, or fragment or variant thereof, as compared tothe half-life of the Therapeutic protein X, or fragment or variantthereof, in an unfused state.
 32. The albumin fusion protein of claim 6,wherein said albumin fusion protein comprises a portion of albuminsufficient to increase the half-life of the Therapeutic protein X, orfragment or variant thereof, as compared to the half-life of theTherapeutic protein X, or fragment or variant thereof, in an unfusedstate.
 33. A method of increasing the half-life of Therapeutic proteinX, or fragment or variant thereof, comprising the step of fusing theTherapeutic protein X, or fragment or variant thereof, to albumin, orfragment or variant thereof, sufficient to increase the half-life of theTherapeutic protein X, or fragment or variant thereof, compared to thehalf-life of the Therapeutic protein X, or fragment or variant thereof,in an unfused state.